cell cycle-dependent expression: Topics by Science.gov

Sample records for cell cycle-dependent expression

Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

PubMed

Cooper, Stephen

2017-11-01

Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.
Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle.

PubMed

Picazo, R A; García Ruiz, J P; Santiago Moreno, J; González de Bulnes, A; Muñoz, J; Silván, G; Lorenzo, P L; Illera, J C

2004-11-01

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.
Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

PubMed

Hu, Shen; Le, Zhang; Krylov, Sergey; Dovichi, Norman J

2003-07-15

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.
Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.
Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390
Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

PubMed

Bogdanow, Boris; Weisbach, Henry; von Einem, Jens; Straschewski, Sarah; Voigt, Sebastian; Winkler, Michael; Hagemeier, Christian; Wiebusch, Lüder

2013-10-22

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.
Cyclin-dependent kinase inhibitor p20 controls circadian cell-cycle timing

PubMed Central

Laranjeiro, Ricardo; Tamai, T. Katherine; Peyric, Elodie; Krusche, Peter; Ott, Sascha; Whitmore, David

2013-01-01

Specific stages of the cell cycle are often restricted to particular times of day because of regulation by the circadian clock. In zebrafish, both mitosis (M phase) and DNA synthesis (S phase) are clock-controlled in cell lines and during embryo development. Despite the ubiquitousness of this phenomenon, relatively little is known about the underlying mechanism linking the clock to the cell cycle. In this study, we describe an evolutionarily conserved cell-cycle regulator, cyclin-dependent kinase inhibitor 1d (20 kDa protein, p20), which along with p21, is a strongly rhythmic gene and directly clock-controlled. Both p20 and p21 regulate the G1/S transition of the cell cycle. However, their expression patterns differ, with p20 predominant in developing brain and peak expression occurring 6 h earlier than p21. p20 expression is also p53-independent in contrast to p21 regulation. Such differences provide a unique mechanism whereby S phase is set to different times of day in a tissue-specific manner, depending on the balance of these two inhibitors. PMID:23569261
Cyclin-dependent kinase inhibitor p20 controls circadian cell-cycle timing.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Peyric, Elodie; Krusche, Peter; Ott, Sascha; Whitmore, David

2013-04-23

Specific stages of the cell cycle are often restricted to particular times of day because of regulation by the circadian clock. In zebrafish, both mitosis (M phase) and DNA synthesis (S phase) are clock-controlled in cell lines and during embryo development. Despite the ubiquitousness of this phenomenon, relatively little is known about the underlying mechanism linking the clock to the cell cycle. In this study, we describe an evolutionarily conserved cell-cycle regulator, cyclin-dependent kinase inhibitor 1d (20 kDa protein, p20), which along with p21, is a strongly rhythmic gene and directly clock-controlled. Both p20 and p21 regulate the G1/S transition of the cell cycle. However, their expression patterns differ, with p20 predominant in developing brain and peak expression occurring 6 h earlier than p21. p20 expression is also p53-independent in contrast to p21 regulation. Such differences provide a unique mechanism whereby S phase is set to different times of day in a tissue-specific manner, depending on the balance of these two inhibitors.
Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.
Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127
PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

PubMed

Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

2001-10-11

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.
A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906
Modeling Bi-modality Improves Characterization of Cell Cycle on Gene Expression in Single Cells

PubMed Central

Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

2014-01-01

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%–17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome. PMID:25032992
Ubiquitin specific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells.

PubMed

Nepal, Saroj; Shrestha, Anup; Park, Pil-Hoon

2015-09-05

Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, respectively in cancer cells. Ubiquitin specific protease-2 (USP-2), a deubiquitinating enzyme, is known to impair proteasome-induced degradation of cyclin D1, a critical cell cycle regulator. Herein, we investigated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expression both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyclin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together, our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regulation by adipokines. Thus, USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

PubMed

Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

2015-07-01

Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.
Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

PubMed Central

Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

2000-01-01

The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660
Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

PubMed

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.
Cell Cycle-Dependent Rho GTPase Activity Dynamically Regulates Cancer Cell Motility and Invasion In Vivo

PubMed Central

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. PMID:24386239
FHL2 regulates cell cycle-dependent and doxorubicin-induced p21Cip1/Waf1 expression in breast cancer cells.

PubMed

Martin, Bernd T; Kleiber, Kai; Wixler, Viktor; Raab, Monika; Zimmer, Brigitte; Kaufmann, Manfred; Strebhardt, Klaus

2007-07-15

The transcriptional cofactor FHL2 interacts with a broad variety of transcription factors and its expression is often deregulated in various types of cancer. Here we analyzed for the first time the molecular function of FHL2 in breast cancer. FHL2 is overexpressed in almost all human mammary carcinoma samples tested but not in normal breast tissues and only low levels of FHL2 expression were present in four premalignant ductal carcinoma in situ (DCIS). Cell cycle analysis revealed an upregulation of endogenous FHL2 towards G2/M in MDA-MB 231 cells and an accelerated G2/M transition when FHL2 expression was suppressed in these cells. In search for G2/M specific target genes regulated by FHL2, we found that expression of the cell cycle inhibitor p21Cip1/Waf1 (hereafter p21) is dependent on FHL2 in MDA-MB 231 breast cancer cells. Downregulation of FHL2 by shRNA abrogated the cell cycle dependent upregulation of p21 as well as the induction of p21 in response to treatment with the DNA damaging agent doxorubicin. FHL2-dependent p21 expression occurs in a p53-independent manner and p21 expression can be downregulated by specific inhibition of mitogen-activated protein kinases (MAPKs), implicating an involvement of MAPK signaling in this regulation. Analysis of FHL2 contribution to the MAPK signaling identified FHL2 as an important downstream effector of MAPKs in breast cancer cells, capable of transactivating endogenous AP1 target genes as well as AP1 dependent reporter genes. Finally, downregulation of FHL2 reduces the ability of MDA-MB 231 cells to form colonies in soft agar, while FHL2 overexpression enhances colony formation of breast cancer cells. Thus, our findings indicate that overexpression of the transcriptional cofactor FHL2 contributes to breast cancer development by mediating transcriptional activation of MAPK target genes known to be involved in cancer progression, such as p21.
ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

PubMed Central

Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

2016-01-01

It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

PM2.5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues.

PubMed

Zhao, Hui; Yang, Biao; Xu, Jia; Chen, Dong-Mei; Xiao, Chun-Ling

2017-06-01

The aim of the current study was to investigate the expression of cell cycle-associated genes induced by fine particulate matter (PM 2.5 ) in lung cancer cell line and tissues. The pulmonary lymph node metastasis cells (H292) were treated with PM 2.5 in vitro. Wistar rats were used to perform an in vivo study. Rats were randomly assigned to experiment and control groups and those in the experiment group were exposed to PM 2.5 once every 15 d, while those in the control group were exposed to normal saline. The cell cycle-associated genes expression was analyzed by real-time PCR. Trachea and lung tissues of rats were processed for scanning electron microscopic (SEM) examinations. Exposure of H292 cells to PM 2.5 dramatically increased the expressions of p53 and cyclin-dependent kinase 2 (CDK2) after 24h of exposure (p<0.01) and markedly increased the expressions of the cell division cycle 2 (Cdc2) and cyclin B after 48h of exposure (p<0.01), while those genes expressions were significantly reduced after 72h of exposure, at which time the expression of p21 was predominant (p<0.01). In vivo studies further demonstrated these results. The results of SEM suggested that both of the trachea and lung tissues were damaged and the degree of damage was time-dependent. In conclusion, PM 2.5 can induce significantly alterations of p53 and CDK2 in the early phase, Cdc2 and cyclin B in mid-term and p21 in long-term exposure. The degree of PM 2.5 -induced damage to the trachea and lung tissue was time-dependent. Copyright © 2017. Published by Elsevier B.V.
Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

PubMed Central

Ruijtenberg, Suzan; van den Heuvel, Sander

2016-01-01

ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227
A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

PubMed

Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

2016-05-01

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

PubMed

Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

2015-05-12

This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.
Induction of S phase cell arrest and caspase activation by polysaccharide peptide isolated from Coriolus versicolor enhanced the cell cycle dependent activity and apoptotic cell death of doxorubicin and etoposide, but not cytarabine in HL-60 cells.

PubMed

Hui, Kenrie Pui-Yan; Sit, Wai-Hung; Wan, Jennifer Man-Fan

2005-07-01

Activation of the cell death program (apoptosis) is a strategy for the treatment of human cancer, and unfortunately a large number of drugs identified as cell cycle-specific agents for killing cancer cells are also toxic to normal cells. The present study demonstrates that the polysaccharide peptide (PSP) extracted from the Chinese medicinal mushroom, Coriolus versicolor, used in combination therapy in China, has the ability to lower the cytotoxicity of certain anti-leukemic drugs via their interaction with cell cycle-dependent and apoptotic pathways. Flow cytometry analysis demonstrated that pre-treatment of PSP (25-100 microg/ml) dose-dependently enhanced the cell cycle perturbation and apoptotic activity of doxorubicin (Doxo) and etoposide (VP-16), but not cytarabine (Ara-C) in human promyelocytic leukemia HL-60 cells. The antagonistic result from combined treatment with Ara-C and PSP may be caused by the removal of HL-60 cells in the G1-S boundary by PSP before exposure to Ara-C. A negative correlation between the increase in apoptotic cell population (pre-G1 peak) with the S-phase cell population expression (R2=0.998), the expression of cyclin E expression (R2=0.872) and caspase 3 activity (R2=0.997) suggests that PSP enhanced the apoptotic machinery of Doxo and VP-16 in a cell cycle-dependent manner and is mediated, at least in part, by the PSP-mediated modulation of the regulatory checkpoint cyclin E and caspase 3. This study is the first to describe the cell cycle mechanistic action of PSP and its interaction with other anticancer agents. Our data support the potential development of PSP as an adjuvant for leukemia treatment, but also imply the importance of understanding its interaction with individual anticancer agents.
CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

PubMed

Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

2006-05-01

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.
Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

PubMed Central

Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

2016-01-01

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731
Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

PubMed

Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

2015-08-14

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth. Copyright © 2015 Elsevier Inc. All rights reserved.
Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.
A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells.

PubMed

Chen, C; Yang, R L

2013-08-01

MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.
Transactivation mediated by B-Myb is dependent on TAF(II)250.

PubMed

Bartusel, Thorsten; Klempnauer, Karl-Heinz

2003-05-15

B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. We have employed the hamster cell line ts13 as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells.
p205, a potential tumor suppressor, inhibits cell proliferation via multiple pathways of cell cycle regulation.

PubMed

Asefa, Benyam; Dermott, Jonathan M; Kaldis, Philipp; Stefanisko, Karen; Garfinkel, David J; Keller, Jonathan R

2006-02-20

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.
Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.
Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.
MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.

PubMed

Shostak, Anton; Ruppert, Bianca; Ha, Nati; Bruns, Philipp; Toprak, Umut H; Eils, Roland; Schlesner, Matthias; Diernfellner, Axel; Brunner, Michael

2016-06-24

The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.
Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

PubMed Central

Scotti, Claudia; Sommi, Patrizia; Pasquetto, Maria Valentina; Cappelletti, Donata; Stivala, Simona; Mignosi, Paola; Savio, Monica; Chiarelli, Laurent Roberto; Valentini, Giovanna; Bolanos-Garcia, Victor M.; Merrell, Douglas Scott; Franchini, Silvia; Verona, Maria Luisa; Bolis, Cristina; Solcia, Enrico; Manca, Rachele; Franciotta, Diego; Casasco, Andrea; Filipazzi, Paola; Zardini, Elisabetta; Vannini, Vanio

2010-01-01

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application. PMID:21085483
Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

PubMed Central

Xie, You-Li; Tao, Wen-Hui; Yang, Ti-Xiong; Qiao, Jian-Guo

2016-01-01

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma. PMID:27698776
ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

PubMed

Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

2016-08-02

ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.
Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis.

PubMed

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.
Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms

PubMed Central

Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang

2014-01-01

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27Kip1, p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27Kip1 at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. PMID:25349217

Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms.

PubMed

Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang; Li, Rongshan

2015-07-01

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. © 2014 by the Society for Experimental Biology and Medicine.
Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression.

PubMed

Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-05-23

Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27(kip-1) increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27(kip-1).
A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

PubMed Central

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer. PMID:23903687
Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

PubMed Central

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2016-01-01

Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301. PMID:27673332
Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells.

PubMed

Jarząb, Agata; Grabarska, Aneta; Kiełbus, Michał; Jeleniewicz, Witold; Dmoszyńska-Graniczka, Magdalena; Skalicka-Woźniak, Krystyna; Sieniawska, Elwira; Polberg, Krzysztof; Stepulak, Andrzej

2014-11-01

The aim of the present study was to determine the effects of osthole on cell proliferation and viability, cell-cycle progression and induction of apoptosis in human laryngeal cancer RK33 and human medulloblastoma TE671 cell lines. Cell viability was measured by means of the MTT method and cell proliferation by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell-cycle progression was determined by flow cytometry, and induction of apoptosis by release of oligonucleosomes to the cytosol. The gene expression was estimated by a quantitative polymerase chain reaction (qPCR) method. High-performance counter-current chromatography (HPCCC) was applied for isolation of osthole from fruits of Mutellina purpurea. Osthole decreased proliferation and cell viability of cancer cells in a dose-dependent manner. The tested compound induced apoptosis, increased the cell numbers in G1 and decreased cell number in S/G2 phases of the cell cycle, differentially regulating CDKN1A and TP53 gene expression depending on cancer cell type. Osthole could be considered as a potential compound for cancer therapy and chemoprevention. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Antitumor Effects of Flavopiridol on Human Uterine Leiomyoma In Vitro and in a Xenograft Model

PubMed Central

Lee, Hyun-Gyo; Baek, Jong-Woo; Shin, So-Jin; Kwon, Sang-Hoon; Cha, Soon-Do; Park, Won-Jin; Chung, Rosa; Choi, Eun-Som; Lee, Gun-Ho

2014-01-01

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21cip/wafl and p27kip1 in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma. PMID:24572052
Nitric oxide is involved in hydrogen gas-induced cell cycle activation during adventitious root formation in cucumber.

PubMed

Zhu, Yongchao; Liao, Weibiao; Niu, Lijuan; Wang, Meng; Ma, Zhanjun

2016-06-28

Adventitious root development is a complex process regulated through a variety of signaling molecules. Hydrogen gas (H2) and nitric oxide (NO), two new signaling molecules are both involved in plant development and stress tolerance. To investigate the mechanism of adventitious root development induced by hydrogen-rich water (HRW), a combination of fluorescence microscopy and molecular approaches was used to study cell cycle activation and cell cycle-related gene expression in cucumber (Cucumis sativus 'Xinchun 4') explants. The results revealed that the effect of HRW on adventitious root development was dose-dependent, with maximal biological responses at 50 % HRW. HRW treatment increased NO content in a time-dependent fashion. The results also indicated that HRW and NO promoted the G1-to-S transition and up-regulated cell cycle-related genes: CycA (A-type cyclin), CycB (B-type cyclin), CDKA (cyclin-dependent kinase A) and CDKB (cyclin-dependent kinase B) expression. Additionally, target genes related to adventitious rooting were up-regulated by HRW and NO in cucumber explants. While, the responses of HRW-induced adventitious root development and increase of NO content were partially blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, NO synthase (NOS)-like enzyme inhibitor N(G) -nitro-L-arginine methylester hydrochloride, or nitrate reductase inhibitors tungstate and NaN3. These chemicals also partially reversed the effect of HRW on cell cycle activation and the transcripts of cell cycle regulatory genes and target genes related adventitious root formation. Together, NO may emerge as a downstream signaling molecule in H2-induced adventitious root organogenesis. Additionally, H2 mediated cell cycle activation via NO pathway during adventitious root formation.
Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

PubMed

Zan, Xinyi; Cui, Fengjie; Li, Yunhong; Yang, Yan; Wu, Di; Sun, Wenjing; Ping, Lifeng

2015-05-01

HEG-5 is a novel polysaccharide-protein purified from the fermented mycelia of Hericium erinaceus CZ-2. The present study aims to investigate the effects of HEG-5 on proliferation, cell cycle and apoptosis of human gastric cancer cells SGC-7901. Here, we first uncover that HEG-5 significantly inhibited the proliferation and colony formation of SGC-7901 cells by promoting apoptosis and cell cycle arrest at S phase. RT-PCR and Western blot analysis suggested that HEG-5 could decrease the expressions of Bcl2, PI3K and AKT1, while increase the expressions of Caspase-8, Caspase-3, p53, CDK4, Bax and Bad. These findings indicated that the Caspase-8/-3-dependent, p53-dependent mitochondrial-mediated and PI3k/Akt signaling pathways involved in the molecular events of HEG-5 induced apoptosis and cell cycle arrest. Thus, our study provides in vitro evidence that HEG-5 may be taken as a potential candidate for treating gastric cancer. Copyright © 2015 Elsevier B.V. All rights reserved.
A Protein Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Is Expressed in Trigeminal Ganglionic Neurons of Latently Infected Cattle and Interacts with Cyclin-Dependent Kinase 2 during Productive Infection

PubMed Central

Jiang, Yunquan; Hossain, Ashfaque; Winkler, Maria Teresa; Holt, Todd; Doster, Alan; Jones, Clinton

1998-01-01

Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807–3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation. PMID:9733854
A protein encoded by the latency-related gene of bovine herpesvirus 1 is expressed in trigeminal ganglionic neurons of latently infected cattle and interacts with cyclin-dependent kinase 2 during productive infection.

PubMed

Jiang, Y; Hossain, A; Winkler, M T; Holt, T; Doster, A; Jones, C

1998-10-01

Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807-3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation.
Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

PubMed

Wang, Tao; Xie, Zi-ping; Huang, Zhan-sen; Li, Hao; Wei, An-yang; Di, Jin-ming; Xiao, Heng-jun; Zhang, Zhi-gang; Cai, Liu-hong; Tao, Xin; Qi, Tao; Chen, Di-ling; Chen, Jun

2015-10-01

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.
Expression of the proliferation marker Ki-67 during early mouse development.

PubMed

Winking, H; Gerdes, J; Traut, W

2004-01-01

In somatic tissues, the mouse Ki-67 protein (pKi-67) is expressed in proliferating cells only. Depending on the stage of the cell cycle, pKi-67 is associated with different nuclear domains: with euchromatin as part of the perichromosomal layer, with centromeric heterochromatin, and with the nucleolus. In gametes, sex-specific expression is evident. Mature MII oocytes contain pKi-67, whereas pKi-67 is not detectable in mature sperm. We investigated the re-establishment of the cell cycle-dependent distribution of pKi-67 during early mouse development. After fertilization, male and female pronuclei exhibited very little or no pKi-67, while polar bodies were pKi-67 positive. Towards the end of the first cell cycle, prophase chromosomes of male and female pronuclei simultaneously got decorated with pKi-67. In 2-cell embryos, the distribution pattern changed, presumably depending on the progress of development of the embryo, from a distribution all over the nucleus to a preferential location in the nucleolus precursor bodies (NPBs). From the 4-cell stage onwards, pKi-67 showed the regular nuclear relocations known from somatic tissues: during mitosis the protein was found covering the chromosome arms as a constituent of the perichromosomal layer, in early G1 it was distributed in the whole nucleus, and for the rest of the cell cycle it was associated with NPBs or with the nucleolus. Copyright 2004 S. Karger AG, Basel
Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonarymore » hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.« less
Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

PubMed Central

Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962
An extensive program of periodic alternative splicing linked to cell cycle progression

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Weatheritt, Robert; Wang, Yang; Blencowe, Benjamin J; Wang, Zefeng

2016-01-01

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control. DOI: http://dx.doi.org/10.7554/eLife.10288.001 PMID:27015110
Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage.

PubMed

Karimian, Ansar; Ahmadi, Yasin; Yousefi, Bahman

2016-06-01

An appropriate control over cell cycle progression depends on many factors. Cyclin-dependent kinase (CDK) inhibitor p21 (also known as p21(WAF1/Cip1)) is one of these factors that promote cell cycle arrest in response to a variety of stimuli. The inhibitory effect of P21 on cell cycle progression correlates with its nuclear localization. P21 can be induced by both p53-dependent and p53-independent mechanisms. Some other important functions attributed to p21 include transcriptional regulation, modulation or inhibition of apoptosis. These functions are largely dependent on direct p21/protein interactions and also on p21 subcellular localizations. In addition, p21 can play a role in DNA repair by interacting with proliferating cell nuclear antigen (PCNA). In this review, we will focus on the multiple functions of p21 in cell cycle regulation, apoptosis and gene transcription after DNA damage and briefly discuss the pathways and factors that have critical roles in p21 expression and activity. Copyright © 2016 Elsevier B.V. All rights reserved.
Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis

PubMed Central

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells. PMID:25674209
Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

PubMed

Bruni, R; Roizman, B

1998-11-01

The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the alpha22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparent Mr of 78,000 and two minor bands with apparent Mrs of 62, 000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.
Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression

PubMed Central

Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-01-01

Background/Aims Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. Methods The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. Results The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27kip-1 increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Conclusions Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27kip-1. PMID:26470770
ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.

PubMed

Chevrollier, Arnaud; Loiseau, Dominique; Gautier, Fabien; Malthièry, Yves; Stepien, Georges

2005-01-01

Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.
Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

PubMed

Yap, W H; Khoo, K S; Lim, S H; Yeo, C C; Lim, Y M

2012-01-15

Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells. Copyright © 2011 Elsevier GmbH. All rights reserved.
[Effect of shikonin, a phytocompound from Lithospermum erythrorhizon, on rat vascular smooth muscle cells proliferation and apoptosis in vitro].

PubMed

Zhang, Zhuo-qi; Cao, Xi-chuan; Zhang, Ling; Zhu, Wen-ling

2005-06-08

To study the anti-proliferation, pro-apoptosis and cell cycle blocking effects of shikonin on rat vascular smooth muscle cell (VSMC) in vitro. VSMCs were primarily cultured by explant method from the thoracic aorta of male SD rats. Shikonin of different concentration, 4, 2, 1, 0.5, 0.25, and 0 micromol/L was added. The cell viability was detected by MTT method. Cell growth curve was drawn by trypan blue exclusion method. (3)H-thymidine incorporation was used to calculate the inhibition rate of DNA synthesis. Flow cytometry was used to detect the cell cycle. Cell apoptosis was observed by fluorescence microscopy. Western blotting was performed to detect the expression of different cell apoptosis and cell cycle regulatory proteins, such as cyclin D(1) and E, proliferating cell nuclear antigen (PCNA), p21(waf1/cip1), p27(kip1), and p53. Compared with control group, shikonin had no obvious cytotoxic effect on cell viability at the concentration of 0.25-1 micromol/L (P > 0.05). While it could inhibit, both time- and dose-dependently, the growth of VSMC, which was predominant of 1 micromol/L at 72 h (1.9 x 10(5)/well vs 5.8 x 10(5)/well, P < 0.05), and DNA synthesis was also significantly inhibited in a time- and dose-dependent manner with inhibition rate varied from 33 to 98% (P < 0.05 or P < 0.01). 1 micromol/L shikonin significantly blocked the cell cycle progression in proliferative VSMC, decreased S, G(2)/M phase (P < 0.05) and increased G(0)/G(1) phase (P < 0.05) to quiescent level with sub-G(1) apoptotic distribution at 48 h (10.9% +/- 0.3%). Shikohin at the concentration of 1-2 micromol/L significantly increased the percentage of apoptotic cells in a time- and dose-dependent manner compared with control group (2.8%-23.7% vs 0.2%-0.4%, P < 0.05), and typical apoptotic nuclear morphological changes were observed. 1 micromol/L shikonin significantly down-regulated cyclin D(1), E and PCNA expression, up-regulated p21(wif1/cip1) expression, and did not obviously influence the p27(kip1) and p53 expression. Shikonin inhibits the proliferation, promotes the apoptosis and blocks cell cycle progression of VSMC. These effects are associated with the expression changes of cell cycle regulatory proteins.
Cell cycle gene expression under clinorotation

NASA Astrophysics Data System (ADS)

Artemenko, Olga

2016-07-01

Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.
15-PGDH/15-KETE plays a role in hypoxia-induced pulmonary vascular remodeling through ERK1/2-dependent PAR-2 pathway.

PubMed

Wei, Liuping; Yu, Xiufeng; Shi, Hengyuan; Zhang, Bo; Lian, Mingming; Li, Jing; Shen, Tingting; Xing, Yan; Zhu, Daling

2014-07-01

We have established that 15-hydroxyeicosatetraenoic acid is an important factor in regulation of pulmonary vascular remodeling (PVR) associated with hypoxia-induced pulmonary hypertension (PH), which is further metabolized by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to form 15-ketoeicosatetraenoic acid (15-KETE). However, the role of 15-PGDH and 15-KETE on PH has not been identified. The purpose of this study was to investigate whether 15-PGDH/15-KETE pathway regulates hypoxia-induced PVR in PH and to characterize the underlying mechanisms. To accomplish this, Immunohistochemistry, Ultra Performance Liquid Chromatography, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed. Our results showed that the levels of 15-PGDH expression and endogenous 15-KETE were drastically elevated in the lungs of humans with PH and hypoxic PH rats. Hypoxia stimulated pulmonary arterial smooth muscle cell (PASMC) proliferation, which seemed to be due to the increased 15-PGDH/15-KETE. 15-PGDH/15-KETE pathway was also capable of stimulating the cell cycle progression and promoting the cell cycle-related protein expression. Furthermore, 15-KETE-promoted cell cycle progression and proliferation in PASMCs depended on protease-activated receptor 2 (PAR-2). ERK1/2 signaling was likely required for 15-PGDH/15-KETE-induced PAR-2 expression under hypoxia. Our study indicates that 15-PGDH/15-KETE stimulates the cell cycle progression and proliferation of PASMCs involving ERK1/2-mediated PAR-2 expression, and contributes to hypoxia-induced PVR. Copyright © 2014 Elsevier Inc. All rights reserved.
Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

PubMed

Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

2017-01-01

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
A Transient Expression of Prospero Promotes Cell Cycle Exit of Drosophila Postembryonic Neurons through the Regulation of Dacapo

PubMed Central

Colonques, Jordi; Ceron, Julian; Reichert, Heinrich; Tejedor, Francisco J.

2011-01-01

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor PROSPERO is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that PROSPERO is not expressed in postmitotic neuronal progeny. Thus, PROSPERO is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. Here we focus on postembryonic neurogenesis and show that the expression of PROSPERO is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, we provide evidence that this expression of PROSPERO in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) DACAPO. These findings imply that PROSPERO, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development. PMID:21552484
Resveratrol induces cell cycle arrest and apoptosis in human eosinophils from asthmatic individuals.

PubMed

Hu, Xin; Wang, Jing; Xia, Yu; Simayi, Mihereguli; Ikramullah, Syed; He, Yuanbing; Cui, Shihong; Li, Shuang; Wushouer, Qimanguli

2016-12-01

Eosinophils exert a number of inflammatory effects through the degranulation and release of intracellular mediators, and are considered to be key effector cells in allergic disorders, including asthma. In order to investigate the regulatory effects of the natural polyphenol, resveratrol, on eosinophils derived from asthmatic individuals, the cell counting Kit‑8 assay and flow cytometry analysis were used to determine cell proliferation and cell cycle progression in these cells, respectively. Cellular apoptosis was detected using annexin V-fluorescein isothiocyanate/propidium iodide double‑staining. The protein expression levels of p53, p21, cyclin‑dependent kinase 2 (CDK2), cyclin A, cyclin E, Bim, B‑cell lymphoma (Bcl)‑2 and Bcl‑2‑associated X protein (Bax) were measured by western blot analysis following resveratrol treatment. The results indicated that resveratrol effectively suppressed the proliferation of eosinophils from asthmatic patients in a concentration‑ and time‑dependent manner. In addition, resveratrol was observed to arrest cell cycle progression in G1/S phase by increasing the protein expression levels of p53 and p21, and concurrently reducing the protein expression levels of CDK2, cyclin A and cyclin E. Furthermore, resveratrol treatment significantly induced apoptosis in eosinophils, likely through the upregulation of Bim and Bax protein expression levels and the downregulation of Bcl‑2 protein expression. These findings suggested that resveratrol may be a potential agent for the treatment of asthma by decreasing the number of eosinophils.
Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

2015-04-01

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21.

PubMed

Gongpan, Pianchou; Lu, Yanting; Wang, Fang; Xu, Yuhui; Xiong, Wenyong

2016-07-02

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.
Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action.

PubMed

Chilampalli, Chandeshwari; Guillermo, Ruth; Zhang, Xiaoying; Kaushik, Radhey S; Young, Alan; Zeman, David; Hildreth, Michael B; Fahmy, Hesham; Dwivedi, Chandradhar

2011-10-20

Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.
Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

PubMed Central

2011-01-01

Background Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. Methods UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Results Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Conclusions Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer. PMID:22014088
Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

PubMed

Poncelet, Luc; Garigliany, Mutien; Ando, Kunie; Franssen, Mathieu; Desmecht, Daniel; Brion, Jean-Pierre

2016-12-16

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.
Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells

PubMed Central

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation. PMID:24695638
Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

PubMed

van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

2014-01-01

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.
PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.
Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway.

PubMed

Yuan, Gangjun; Chen, Xin; Liu, Zhuowei; Wei, Wensu; Shu, Qinghai; Abou-Hamdan, Hussein; Jiang, Lijuan; Li, Xiangdong; Chen, Rixin; Désaubry, Laurent; Zhou, Fangjian; Xie, Dan

2018-02-07

Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored. FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms. FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent. Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.
The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.
The DREAM complex: Master coordinator of cell cycle dependent gene expression

PubMed Central

Sadasivam, Subhashini; DeCaprio, James A.

2014-01-01

Preface The dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM) complex provides a previously unsuspected unifying role in the cell cycle by directly linking p130, p107, E2F, BMYB and FOXM1. DREAM mediates gene repression during G0 and coordinates periodic gene expression with peaks during G1/S and G2/M. Perturbations in DREAM regulation shift the balance from quiescence towards proliferation and contribute to increased mitotic gene expression levels frequently observed in cancers with poor prognosis. PMID:23842645
[Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

PubMed

Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

2017-12-01

Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

Changes in endometrial natural killer cell expression of CD94, CD158a and CD158b are associated with infertility.

PubMed

McGrath, Emma; Ryan, Elizabeth J; Lynch, Lydia; Golden-Mason, Lucy; Mooney, Eoghan; Eogan, Maeve; O'Herlihy, Colm; O'Farrelly, Cliona

2009-04-01

Cycle-dependent fluctuations in natural killer (NK) cell populations in endometrium and circulation may differ, contributing to unexplained infertility. NK cell phenotypes were determined by flow cytometry in endometrial biopsies and matched blood samples. While circulating and endometrial T cell populations remained constant throughout the menstrual cycle in fertile and infertile women, circulating NK cells in infertile women increased during the secretory phase. However, increased expression of CD94, CD158b (secretory phase), and CD158a (proliferative phase) by endometrial NK cells from infertile women was observed. These changes were not reflected in the circulation. In infertile women, changes in circulating NK cell percentages are found exclusively during the secretory phase and not in endometrium; cycle-related changes in NK receptor expression are observed only in infertile endometrium. While having exciting implications for understanding NK cell function in fertility, our data emphasize the difficulty in attaching diagnostic or prognostic significance to NK cell analyses in individual patients.
[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

PubMed Central

Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

2006-01-01

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513
FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms*

PubMed Central

Czymai, Tobias; Viemann, Dorothee; Sticht, Carsten; Molema, Grietje; Goebeler, Matthias; Schmidt, Marc

2010-01-01

FOXO transcription factors represent targets of the phosphatidylinositol 3-kinase/protein kinase B survival pathway controlling important biological processes, such as cell cycle progression, apoptosis, vascular remodeling, stress responses, and metabolism. Recent studies suggested the existence of alternative mechanisms of FOXO-dependent gene expression beyond classical binding to a FOXO-responsive DNA-binding element (FRE). Here we analyzed the relative contribution of those mechanisms to vascular function by comparing the transcriptional and cellular responses to conditional activation of FOXO3 and a corresponding FRE-binding mutant in human primary endothelial cells. We demonstrate that FOXO3 controls expression of vascular remodeling genes in an FRE-dependent manner. In contrast, FOXO3-induced cell cycle arrest and apoptosis occurs independently of FRE binding, albeit FRE-dependent gene expression augments the proapoptotic response. These findings are supported by bioinformatical analysis, which revealed a statistical overrepresentation of cell cycle regulators and apoptosis-related genes in the group of co-regulated genes. Molecular analysis of FOXO3-induced endothelial apoptosis excluded modulators of the extrinsic death receptor pathway and demonstrated important roles for the BCL-2 family members BIM and NOXA in this process. Although NOXA essentially contributed to FRE-dependent apoptosis, BIM was effectively induced in the absence of FRE-binding, and small interfering RNA-mediated BIM depletion could rescue apoptosis induced by both FOXO3 mutants. These data suggest BIM as a critical cell type-specific mediator of FOXO3-induced endothelial apoptosis, whereas NOXA functions as an amplifying factor. Our study provides the first comprehensive analysis of alternatively regulated FOXO3 targets in relevant primary cells and underscores the importance of such genes for endothelial function and integrity. PMID:20123982
Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

PubMed

Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

2017-08-01

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.
HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

PubMed

Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

2018-04-01

Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1 particle production but, unexpectedly, are completely resistant to virus-induced cytopathic effects. We mapped these effects to the viral accessory protein Vif, which induces a prolonged G 2 /M cell cycle arrest followed by apoptosis in human cells. Combined, our results indicate that one or more additional human-specific cofactors govern HIV-1's capacity to modulate the cell cycle, with potential relevance to viral pathogenesis in people and existing animal models. Copyright © 2018 American Society for Microbiology.
Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions

PubMed Central

Sunter, Jack D.; Benz, Corinna; Andre, Jane; Whipple, Sarah; McKean, Paul G.; Gull, Keith; Ginger, Michael L.; Lukeš, Julius

2015-01-01

ABSTRACT The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms. PMID:26148511
Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.

PubMed

Wang, Xueju; Dasari, Surendra; Nowakowski, Grzegorz S; Lazaridis, Konstantinos N; Wieben, Eric D; Kadin, Marshall E; Feldman, Andrew L; Boddicker, Rebecca L

2017-04-18

Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.
Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner.

PubMed

Chen, Yi-Hao; Chen, Ching-Long; Lu, Da-Wen; Liang, Chang-Min; Tai, Ming-Cheng; Chen, Jiann-Torng

2016-01-01

The objective of this study was to evaluate the effects of silibinin on cell proliferation in platelet-derived growth factor (PDGF)-treated human Tenon's fibroblasts (HTFs). The effect of silibinin on cell proliferation in PDGF-treated HTFs was determined by examining the expression of proliferating cell nuclear antigen (PCNA) and performing WST-1 assays. Cell cycle progression was evaluated using flow cytometry. The related cyclins and cyclin-dependent kinases (CDKs) were also analyzed using western blot. A modified rat trabeculectomy model was established to evaluate the effect of silibinin on cell proliferation in vivo. Western blot analysis was carried out to determine the effect of silibinin on the expression of PDGF receptor and on the downstream signaling pathways regulated by PDGF receptor. PDGF elevated the expression of PCNA in HTFs, and this elevation was inhibited by silibinin. The inhibitory effect of silibinin on cell proliferation was also confirmed via WST-1 assay. PDGF-stimulated cell cycle in HTFs was delayed by silibinin, and the related cyclin D1 and CDK4 were also suppressed by silibinin. In the rat model of trabeculectomy, silibinin reduced the expression of PCNA at the site of blebs in vivo. The effects of silibinin on PDGF-stimulated HTFs were mediated via the downregulation of PDGF receptor-regulated signaling pathways, such as ERKs and STATs, which may be partially caused by the downregulation of N-glycosylation of PDGF receptor beta (PDGFRβ). The effect of silibinin on modulation of N-glycosylation of PDGFRβ was mediated in a proteasome-dependent manner. Silibinin inhibited cell proliferation and delayed cell cycle progression in PDGF-treated HTFs in vitro. PDGF also modulated the process of N-glycosylation of the PDGFRβ in a proteasome-dependent manner. Our findings suggest that silibinin has potential therapeutic applications in glaucoma filtering surgery.
Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner

PubMed Central

Chen, Yi-Hao; Chen, Ching-Long; Lu, Da-Wen; Liang, Chang-Min; Tai, Ming-Cheng; Chen, Jiann-Torng

2016-01-01

The objective of this study was to evaluate the effects of silibinin on cell proliferation in platelet-derived growth factor (PDGF)-treated human Tenon's fibroblasts (HTFs). The effect of silibinin on cell proliferation in PDGF-treated HTFs was determined by examining the expression of proliferating cell nuclear antigen (PCNA) and performing WST-1 assays. Cell cycle progression was evaluated using flow cytometry. The related cyclins and cyclin-dependent kinases (CDKs) were also analyzed using western blot. A modified rat trabeculectomy model was established to evaluate the effect of silibinin on cell proliferation in vivo. Western blot analysis was carried out to determine the effect of silibinin on the expression of PDGF receptor and on the downstream signaling pathways regulated by PDGF receptor. PDGF elevated the expression of PCNA in HTFs, and this elevation was inhibited by silibinin. The inhibitory effect of silibinin on cell proliferation was also confirmed via WST-1 assay. PDGF-stimulated cell cycle in HTFs was delayed by silibinin, and the related cyclin D1 and CDK4 were also suppressed by silibinin. In the rat model of trabeculectomy, silibinin reduced the expression of PCNA at the site of blebs in vivo. The effects of silibinin on PDGF-stimulated HTFs were mediated via the downregulation of PDGF receptor-regulated signaling pathways, such as ERKs and STATs, which may be partially caused by the downregulation of N-glycosylation of PDGF receptor beta (PDGFRβ). The effect of silibinin on modulation of N-glycosylation of PDGFRβ was mediated in a proteasome-dependent manner. Silibinin inhibited cell proliferation and delayed cell cycle progression in PDGF-treated HTFs in vitro. PDGF also modulated the process of N-glycosylation of the PDGFRβ in a proteasome-dependent manner. Our findings suggest that silibinin has potential therapeutic applications in glaucoma filtering surgery. PMID:28030611
Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

PubMed

Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

2014-01-01

Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.
A high-resolution transcriptome map of cell cycle reveals novel connections between periodic genes and cancer

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Gomez, Nicholas; Jha, Deepak Kumar; Davis, Ian; Wang, Zefeng

2016-01-01

Progression through the cell cycle is largely dependent on waves of periodic gene expression, and the regulatory networks for these transcriptome dynamics have emerged as critical points of vulnerability in various aspects of tumor biology. Through RNA-sequencing of human cells during two continuous cell cycles (>2.3 billion paired reads), we identified over 1 000 mRNAs, non-coding RNAs and pseudogenes with periodic expression. Periodic transcripts are enriched in functions related to DNA metabolism, mitosis, and DNA damage response, indicating these genes likely represent putative cell cycle regulators. Using our set of periodic genes, we developed a new approach termed “mitotic trait” that can classify primary tumors and normal tissues by their transcriptome similarity to different cell cycle stages. By analyzing >4 000 tumor samples in The Cancer Genome Atlas (TCGA) and other expression data sets, we found that mitotic trait significantly correlates with genetic alterations, tumor subtype and, notably, patient survival. We further defined a core set of 67 genes with robust periodic expression in multiple cell types. Proteins encoded by these genes function as major hubs of protein-protein interaction and are mostly required for cell cycle progression. The core genes also have unique chromatin features including increased levels of CTCF/RAD21 binding and H3K36me3. Loss of these features in uterine and kidney cancers is associated with altered expression of the core 67 genes. Our study suggests new chromatin-associated mechanisms for periodic gene regulation and offers a predictor of cancer patient outcomes. PMID:27364684
The Expression Pattern of the Cell Cycle Inhibitor p19INK4d by Progenitor Cells of the Rat Embryonic Telencephalon and Neonatal Anterior Subventricular Zone

PubMed Central

Coskun, Volkan; Luskin, Marla B.

2014-01-01

In this study we investigated whether the pattern of expression of the cyclin-dependent kinase inhibitor p19INK4d by the unique progenitor cells of the neonatal anterior subventricular zone (SVZa) can account for their ability to divide even though they express phenotypic characteristics of differentiated neurons. p19INK4d was chosen for analysis because it usually acts to block permanently the cell cycle at the G1 phase. p19INK4d immunoreactivity and the incorporation of bromodeoxyuridine (BrdU) by SVZa cells were compared with that of the more typical progenitor cells of the prenatal telencephalic ventricular zone. In the developing telencephalon, p19INK4d is expressed by postmitotic cells and has a characteristic perinuclear distribution depending on the laminar position and state of differentiation of a cell. Moreover, the laminar-specific staining of the developing cerebral cortex revealed that the ventricular zone (VZ) is divided into p19INK4d(+) and p19INK4d(−) sublaminae, indicating that the VZ has a previously unrecognized level of functional organization. Furthermore, the rostral migratory stream, traversed by the SVZa-derived cells, exhibits an anteriorhigh–posteriorlow gradient of p19INK4d expression. On the basis of the p19INK4d immunoreactivity and BrdU incorporation, SVZa-derived cells appear to exit and reenter the cell cycle successively. Thus, in contrast to telencephalic VZ cells, SVZa cells continue to undergo multiple rounds of division and differentiation before becoming postmitotic. PMID:11312294
Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation1[OPEN

PubMed Central

Yu, Peng; Eggert, Kai; von Wirén, Nicolaus; Li, Chunjian; Hochholdinger, Frank

2015-01-01

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box proteinS-Phase Kinase-Associated Protein 2B-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses. PMID:26198256
APELA promotes tumour growth and cell migration in ovarian cancer in a p53-dependent manner.

PubMed

Yi, Yuyin; Tsai, Shu-Huei; Cheng, Jung-Chien; Wang, Evan Y; Anglesio, Michael S; Cochrane, Dawn R; Fuller, Megan; Gibb, Ewan A; Wei, Wei; Huntsman, David G; Karsan, Aly; Hoodless, Pamela A

2017-12-01

APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA. Copyright © 2017 Elsevier Inc. All rights reserved.
SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chetty, Chandramu; Dontula, Ranadheer; Ganji, Purnachandra Nagaraju

2012-01-13

Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reductionmore » in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.« less
Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways.

PubMed

Zhao, L M; Pang, A X

2017-01-16

Iodine-131 (131I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following 131I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with 131I. They were then assessed for 131I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and 131I or with a NF-κB inhibitor of BMS-345541 and 131I, non-transfected SW579 cells were assessed in JNK/NFκB pathways. It was observed that 131I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G0/G1 phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and 131I, the non-transfected SW579 cell lines significantly inhibited JNK pathway, NF-κB pathway and the expression of BTG2. However, when treated with BMS-345541 and 131I, only the NF-κB pathway was suppressed. 131I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF-κB pathways.
Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

PubMed Central

Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

2012-01-01

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631
S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

PubMed Central

Khammanivong, Ali; Wang, Chengxing; Sorenson, Brent S.; Ross, Karen F.; Herzberg, Mark C.

2013-01-01

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches. PMID:23874958
Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells.

PubMed

Li, Xiaotong; Xie, Hongqing; Chen, Yajie; Lang, Mingzi; Chen, Yuyin; Shi, Liangen

2018-03-28

Silkworm pupae ( Bombyx mori ) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.
Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

PubMed

Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

2013-07-01

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

PubMed

Wang, Gangduo; Wang, Jianling; Khan, M Firoze

2017-09-01

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.
Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

PubMed Central

Guha, Gunjan; Liang, Xiaobo; Kulesz-Martin, Molly F.; Mahmud, Taifo; Indra, Arup Kumar; Ganguli-Indra, Gitali

2015-01-01

Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action. PMID:25938491
Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity.

PubMed

Duncan, Robin E; Lau, Dominic; El-Sohemy, Ahmed; Archer, Michael C

2004-11-01

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.
Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

PubMed

Lange, S; Viergutz, T; Simkó, M

2004-10-01

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.
TOFA suppresses ovarian cancer cell growth in vitro and in vivo.

PubMed

Li, Shu; Qiu, Lihua; Wu, Buchu; Shen, Haoran; Zhu, Jing; Zhou, Liang; Gu, Liying; Di, Wen

2013-08-01

A characteristic feature of cancer cells is the activation of de novo fatty acid synthesis. Acetyl‑CoA carboxylase (ACC) is a key enzyme in fatty acid synthesis, accelerating the reaction that carboxylates cytosolic acetyl‑CoA to form malonyl‑CoA. ACC is highly expressed in several types of human cancer and is important in breast and prostate cancer cell growth. The aim of the present study was to investigate the effects of 5‑tetradecyloxy‑2‑furoic acid (TOFA), an allosteric inhibitor of ACC, on the proliferation and cell cycle progression of the ovarian cancer cell lines COC1 and COC1/DDP. TOFA was found to be cytotoxic to COC1 and COC1/DDP cells with a 50% inhibitory concentration (IC50) of ~26.1 and 11.6 µg/ml, respectively. TOFA inhibited the proliferation of the cancer cells examined in a time‑ and dose‑dependent manner, arrested the cells in the G0/G1 cell cycle phase and induced apoptosis. The expression of the cell cycle regulating proteins cyclin D1 and cyclin-dependent kinase (CDK) 4, as well as the expression of the apoptosis‑related proteins caspase‑3 and Bcl‑2, were detected by western blot analysis. Cyclin D1, CDK4 and Bcl‑2 protein expression was inhibited by TOFA, while caspase‑3 was cleaved and activated. To the best of our knowledge, the present study demonstrated for the first time that TOFA inhibits COC1/DDP cell growth in ovarian tumor mouse xenografts. By inhibiting ACC, TOFA may be a promising small molecule agent for ovarian cancer therapy.
Inhibitory effect of turmeric curcuminoids on FLT3 expression and cell cycle arrest in the FLT3-overexpressing EoL-1 leukemic cell line.

PubMed

Tima, Singkome; Ichikawa, Hideki; Ampasavate, Chadarat; Okonogi, Siriporn; Anuchapreeda, Songyot

2014-04-25

Leukemia is a hematologic malignancy with a frequent incidence and high mortality rate. Previous studies have shown that the FLT3 gene is overexpressed in leukemic blast cells, especially in acute myeloid leukemia. In this study, a commercially available curcuminoid mixture (1), pure curcumin (2), pure demethoxycurcumin (3), and pure bisdemethoxycurcumin (4) were investigated for their inhibitory effects on cell growth, FLT3 expression, and cell cycle progression in an FLT3-overexpressing EoL-1 leukemic cell line using an MTT assay, Western blotting, and flow cytometry, respectively. The mixture (1) and compounds 2-4 demonstrated cytotoxic effects with IC50 values ranging from 6.5 to 22.5 μM. A significant decrease in FLT3 protein levels was found after curcuminoid treatment with IC20 doses, especially with mixture 1 and compound 2. In addition, mixture 1 and curcumin (2) showed activity on cell cycle arrest at the G0/G1 phase and decreased the FLT3 and STAT5A protein levels in a dose-dependent manner. Compound 2 demonstrated the greatest potential for inhibiting cell growth, cell cycle progression, and FLT3 expression in EoL-1 cells. This investigation has provided new findings regarding the effect of turmeric curcuminoids on FLT3 expression in leukemic cells.
Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer.

PubMed

Madhu Krishna, B; Chaudhary, Sanjib; Mishra, Dipti Ranjan; Naik, Sanoj K; Suklabaidya, S; Adhya, A K; Mishra, Sandip K

2018-05-30

Breast cancer (BC) is highly heterogeneous with ~ 60-70% of estrogen receptor positive BC patient's response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERRβ in breast cancer. Estrogen related receptor β (ERRβ) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERRβ is a direct target of ERα, we investigated the expression of ERRβ in short hairpin ribonucleic acid knockdown of ERα breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ERα by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERRβ in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERRβ with survival in breast cancer patients. Tissue microarray (TMA) analysis showed that ERRβ is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER + ve breast tumors and cell lines showed a significant expression of ERRβ compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERRβ and it was ERα dependent. Mechanistic analyses indicate that ERα directly targets ERRβ through estrogen response element and ERRβ also mediates cell cycle regulation through p18, p21 cip and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERRβ promoter activity in ectopically co-expressed ERα and ERRβ breast cancer cell lines. Fluorescence-activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERRβ overexpressed MCF7 cells. Furthermore, ERRβ expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERRβ in breast cancer cells which provide a potential avenue to target ERRβ signaling pathway in breast cancer. Our results indicate that ERRβ is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERRβ could be therapeutic target for the treatment of breast cancer.
[Effect of taspine derivatives on human liver cancer SMMC7721].

PubMed

Zhang, Yan-min; Wang, Nan; Dai, Bing-ling; He, Lang-chong

2011-07-01

To analyse the inhibition effect of taspine derivatives on human Liver cancer SMMC7721 cell and its mechanism. The effects of five taspine derivatives on SMMC7721 cell growth were determined by MTT. The flow cytometry was used to determine the cell cycle. The effects of Tas-D1 on the EGF and VEGF in SMMC7721 cell were determined by ELISA. The mRNA level of EGF and VEGF in SMMC7721 cell was determined by RT-PCR. The MTT assay demonstrated that the taspine derivative Tas-D1 significantly inhibited the growth of SMMC7721 cell in a dose-dependent manner. Cell was stopped at S phase by Tas-D1. Tas-D1 inhibited the expression of EGF and VEGF and their mRNA in a dose-dependent manner (P<0.05). The taspine derivative Tas-D1 can inhibit the growth of human Liver cancer SMMC7721 cell and change cell cycle, which may be related to the inhibition of EGF and VEGF expression.
Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

PubMed Central

Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

2009-01-01

High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226
Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

PubMed

Diao, Lingyun; Wang, Shengying; Sun, Zhiguang

2018-01-01

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. LncRNA GAPLINC has been reported to be increased in gastric cancer (GC) tissues. Real-time PCR assays were used to measure expressions of GAPLINC, miR-378, and MAPK1 mRNA. Western blot assays were employed to examine MAPK1 protein expression. Cell proliferation and cell cycle were measured by CCK-8 and propidium iodide-detection assays, respectively. The interaction between GAPLINC and miR-378 was confirmed by site-directed mutagenesis and luciferase assays. Luciferase assays were also used to study whether GAPLINC was able to act as a molecular sponge of miR-378 to modulate MAPK1 expression. The lncRNA GAPLINC expression was upregulated and positively correlated with MAPK1 expression in gastric cancer tissues and cells. Additionally, lncRNA GAPLINC promoted the expression of MAPK1 and the enhancement of GC cell proliferation and cell cycle progression by LncRNA GAPLINC was dependent on MAPK1 in vitro and in vivo. Consequently, we found that miR-378 expression was inversely correlated with GAPLINC expression in GC tissues and cells. miR-378 could directly bind to GAPLINC and decreased GAPLINC expression, thus reducing MAPK1 expression. Furthermore, overexpression of miR-378 inhibited MAPK1 expression, cell proliferation, and cell cycle progression of gastric cancer cells, while these effects were abrogated by upregulating lncRNA GAPLINC expression. Taken together, lncRNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.
DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 daysmore » of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.« less
The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

PubMed

Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.
C. elegans STK39/SPAK ortholog-mediated inhibition of ClC anion channel activity is regulated by WNK-independent ERK kinase signaling

PubMed Central

Falin, Rebecca A.; Miyazaki, Hiroaki

2011-01-01

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl− cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes. PMID:21160027
Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells

PubMed Central

Li, Fanni; Dong, Xiwen; Lin, Peng; Jiang, Jianli

2018-01-01

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR. PMID:29360760
Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

PubMed Central

Niculescu, Mihai D.; Yamamuro, Yutaka; Zeisel, Steven H.

2006-01-01

Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G1/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses. PMID:15147518
Galangin Induces Apoptosis in MCF-7 Human Breast Cancer Cells Through Mitochondrial Pathway and Phosphatidylinositol 3-Kinase/Akt Inhibition.

PubMed

Liu, Dan; You, Pengtao; Luo, Yan; Yang, Min; Liu, Yanwen

2018-06-07

The study aimed to investigate the molecular mechanism of inhibition of proliferation and apoptosis induction by galangin against MCF-7 human breast cancer cells. Cell Counting Kit-8 assay was used to assess cell viability and flow cytometry was used to detect cell apoptosis. The expression level of apoptosis-related proteins (cleaved-caspase-9, cleaved-caspase-8, cleaved-caspase-3, Bad, cleaved-Bid, Bcl-2, Bax, p-phosphatidylinositol 3-kinase [PI3K], and p-Akt) and cell cycle-related proteins (cyclin D3, cyclin B1, cyclin-dependent kinases CDK1, CDK2, CDK4, p21, p27, p53) were evaluated by Western blotting. Galangin increased the expression of Bax and decreased the expression of Bcl-2 in a concentration-dependent manner, inhibited cell viability, and induced apoptosis. Meanwhile, the expression of cleavage of caspase-9, caspase-8, caspase-3, Bid, and Bad increased significantly while the expression of p-PI3K and p-Akt proteins decreased. In addition, the protein levels of cyclin D3, cyclin B1, CDK1, CDK2, and CDK4 were downregulated while the expression levels of p21, p27, and p53 were upregulated significantly. Galangin could suppress the viability of MCF-7 cells and induce cell apoptosis via the mitochondrial pathway and PI3K/Akt inhibition as well as cell cycle arrest. © 2018 S. Karger AG, Basel.
Molecular Medicine II: Hormone Dependent Cancers

DTIC Science & Technology

2005-04-01

multimode, live-cell videomicroscopy - we are analyzing the cycles of centrosome amplification in living S-phase arrested cells. Finally, we will directly...living cells by time-lapse multi-mode videomicroscopy . The key construct for our work is the generation of a mammalian somatic cell line stably expressing
Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

PubMed

Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles

2012-09-01

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

PubMed

Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

2016-05-01

Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.
A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts.

PubMed

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-09-15

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation.

[Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

PubMed

Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

2013-05-01

To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.
Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujii, Seiko; Division of Maxillofacial Surgery, Kyushu Dental University; Okinaga, Toshinori

2013-05-10

Highlights: •Novel histone deacetylase inhibitor Ky-2, remarkably inhibits myeloma cell growth. •Ky-2 demonstrates no cytotoxicity against normal lymphocytic cells. •Ky-2 induces cell cycle arrest through the cell cycle-associated proteins. •Ky-2 induces Bcl-2-inhibitable apoptosis through a caspase-dependent cascade. -- Abstract: Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. Materials and methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viabilitymore » was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst’s staining was used to detect apoptotic cells. Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.« less
GENE EXPRESSION PROFILING OF HUMAN LIVER CARCINOMA (HepG2) CELLS EXPOSED TO THE MARINE TOXIN OKADAIC ACID

PubMed Central

Fieber, Lynne A.; Greer, Justin B.; Guo, Fujiang; Crawford, Douglas C.; Rein, Kathleen S.

2012-01-01

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis. PMID:23172983
High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui

The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovariesmore » in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27{sup Kip1} and p21{sup Cip1}, were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure. - Highlights: • HFD induced-obesity leads to abnormal ovarian morphology. • HFD induced-obesity triggers excessive apoptosis in the ovary. • HFD induced-obesity up-regulates cell cycle inhibitors p21{sup Cip1} and p27{sup Kip1} in the ovary. • HFD induced-obesity causes cell cycle arrest in the ovary.« less
The critical role of p16/Rb pathway in the inhibition of GH3 cell cycle induced by T-2 toxin.

PubMed

Fatima, Zainab; Guo, Pu; Huang, Deyu; Lu, Qirong; Wu, Qinghua; Dai, Menghong; Cheng, Guyue; Peng, Dapeng; Tao, Yanfei; Ayub, Muhammad; Ul Qamar, Muhammad Tahir; Ali, Muhammad Waqar; Wang, Xu; Yuan, Zonghui

2018-05-01

T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells. Copyright © 2018 Elsevier B.V. All rights reserved.
PRMT5 is essential for the eIF4E-mediated 5′-cap dependent translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Ji-Hong; Lee, Yoon-Mi; Lee, Gibok

2014-10-03

Highlights: • PRMT5 participates in syntheses of HIF-1α, c-Myc and cyclin D1 proteins. • PRMT5 promotes the 5′-cap dependent translation. • PRMT5 is required for eIF4E binding to mRNA 5′-cap. • PRMT5 is essential for eIF4E-dependent cell proliferation. - Abstract: It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5′-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions aremore » also tightly regulated in 5′-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA–protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5′-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5′-cap dependent translation of proteins essential for proliferation and survival.« less
Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850
Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. PMID:28515305
Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.
Proteomic analysis of the bacterial cell cycle

PubMed Central

Grünenfelder, Björn; Rummel, Gabriele; Vohradsky, Jiri; Röder, Daniel; Langen, Hanno; Jenal, Urs

2001-01-01

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. PMID:11287652
Regulation of steroid hormone receptors and coregulators during the cell cycle highlights potential novel function in addition to roles as transcription factors

PubMed Central

Zheng, Yingfeng; Murphy, Leigh C.

2016-01-01

Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M. PMID:26778927
Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes

PubMed Central

Czerednik, Anna; Busscher, Marco; Bielen, Bram A.M.; Wolters-Arts, Mieke; de Maagd, Ruud A.; Angenent, Gerco C.

2012-01-01

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation. CDKA1 is expressed in the pericarp throughout development, but is strongly up-regulated in the outer pericarp cell layers at the end of the growth period, when CDKB gene expression has ceased. Overexpression of the CDKB genes at later stages of development and the down-regulation of CDKA1 result in a very similar fruit phenotype, showing a reduction in the number of cell layers in the pericarp and alterations in the desiccation of the fruits. Expression studies revealed that CDKA1 is down-regulated by the expression of CDKB1/2 in CDKB1 and CDKB2 overexpression mutants, suggesting opposite roles for these types of CDK proteins in tomato pericarp development. PMID:22282536
Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging.

PubMed

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

2017-03-04

We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G 2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression.
Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging

PubMed Central

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2017-01-01

ABSTRACT We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression. PMID:27715464
Cyclin-dependent kinase inhibitor flavopiridol promotes remyelination in a cuprizone induced demyelination model

PubMed Central

Mi, Guiyun; Gao, Yunyun; Liu, Shuai; Ye, Enmao; Li, Yanyan; Jin, Xiao; Yang, Hongju; Yang, Zheng

2016-01-01

ABSTRACT The cuprizone (CPZ) model has been widely used for the studies of de-and remyelination. The CPZ-exposed mice show oligodendrocyte precursor cells (OPCs) increase and mature oligodendrocytes decrease, suggesting an imbalance between proliferation and differentiation of OPCs. In the first experiment of this study, we examined the expression of cell cycle related genes in brains of mice following CPZ administration for 5 weeks by means of microarray assay. In addition, we performed a double labeling of BrdU and Ki-67 to calculate cell cycle exit index in the mice. Our results showed that CPZ administration up-regulated the expression of 16 cell cycle related genes, but down-regulated the expression of only one in the prefrontal cortex (PFC) of mice compared to control group. The treatment inhibited potential precursor cells exit from cell cycle. In the second experiment, we evaluated effects of a CDK inhibitor flavopiridol (FLA) on CPZ-induced neuropathological changes and spatial working memory impairment in mice.FLA treatment for one week effectively attenuated the CPZ-induced increases in NG2 positive cells, microglia and astrocytes, alleviated the concurrent mature oligodendrocyte loss and myelin breakdown, and improved spatial working memory deficit in the CPZ-exposed mice. These results suggest that CPZ-induced neuropathological changes involve in dysregulation of cell cycle related genes. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition. PMID:27580304
1-(2-Hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells.

PubMed

Tsai, Jie-Heng; Hsu, Li-Sung; Huang, Hsiu-Chen; Lin, Chih-Li; Pan, Min-Hsiung; Hong, Hui-Mei; Chen, Wei-Jen

2016-08-05

The natural agent, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB), has been reported to have growth inhibitory effects on several human cancer cells. However, the role of HMDB in cervical cancer remains unclear. Herein, we found that HMDB dose- and time-dependently inhibited growth of HeLa cervical cancer cells, accompanied with G1 cell cycle arrest. HMDB decreased protein expression of cyclins D1/D3/E and cyclin-dependent kinases (CDKs) 2/4/6 and reciprocally increased mRNA and protein levels of CDK inhibitors (p15, p16, p21, and p27), thereby leading to the accumulation of hypophosphorylated retinoblastoma (Rb) protein. HMDB also triggered the accumulation of acidic vesicles and formation of microtubule-associated protein-light chain 3 (LC3), followed by increased expression of LC3 and Beclin-1 and decreased expression of p62, suggesting that HMDB triggered autophagy in HeLa cells. Meanwhile, suppression of the expression of survivin and Bcl-2 implied that HMDB-induced autophagy is tightly linked to apoptosis. Exploring the action mechanism, HMDB induced autophagy via the modulation of AMP-activated protein kinase (AMPK) and mTOR signaling pathway rather than the class III phosphatidylinositol 3-kinase pathway. These results suggest that HMDB inhibits HeLa cell growth by eliciting a G1 arrest through modulation of G1 cell cycle regulators and by concomitantly inducing autophagy through the mediation of AMPK-mTOR and Akt-mTOR pathways, and may be a promising antitumor agent against cervical cancer.
ATM-dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

PubMed Central

Zhou, Tong; Chou, Jeff; Zhou, Yingchun; Simpson, Dennis A.; Cao, Feng; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

2013-01-01

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. PMID:17699107
Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiao, Lan; Department of Pharmaceutical Sciences, Jilin University, Changchun 130021; Paul, Pritha

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated thatmore » GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.« less
Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

NASA Astrophysics Data System (ADS)

Cariveau, Mickael J.

2005-07-01

Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.
Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.
The BMI1 inhibitor PTC-209 is a potential compound to halt cellular growth in biliary tract cancer cells

PubMed Central

Mayr, Christian; Wagner, Andrej; Loeffelberger, Magdalena; Bruckner, Daniela; Jakab, Martin; Berr, Frieder; Di Fazio, Pietro; Ocker, Matthias; Neureiter, Daniel; Pichler, Martin; Kiesslich, Tobias

2016-01-01

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and is up-regulated in biliary tract cancer (BTC), contributing to aggressive clinical features. In this study we investigated the cytotoxic effects of PTC-209, a recently developed inhibitor of BMI1, in BTC cells. PTC-209 reduced overall viability in BTC cell lines in a dose-dependent fashion (0.04 - 20 μM). Treatment with PTC-209 led to slightly enhanced caspase activity and stop of cell proliferation. Cell cycle analysis revealed that PTC-209 caused cell cycle arrest at the G1/S checkpoint. A comprehensive investigation of expression changes of cell cycle-related genes showed that PTC-209 caused significant down-regulation of cell cycle-promoting genes as well as of genes that contribute to DNA synthesis initiation and DNA repair, respectively. This was accompanied by significantly elevated mRNA levels of cell cycle inhibitors. In addition, PTC-209 reduced sphere formation and, in a cell line-dependent manner, aldehyde dehydrogease-1 positive cells. We conclude that PTC-209 might be a promising drug for future in vitro and in vivo studies in BTC. PMID:26623561
A novel DLX3-PKC integrated signaling network drives keratinocyte differentiation.

PubMed

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-Wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-04-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3-PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.
A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

PubMed Central

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-01-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis. PMID:28186503
Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

PubMed Central

Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

2016-01-01

In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015
HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

PubMed

Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

2015-01-01

Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.
Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

PubMed

Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

2014-10-01

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.
A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Bryan E.; Patricio, Juliana Rotelli; Program in Biotechnology, University of Sao Paulo

2006-10-06

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for thismore » factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.« less
Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium.

PubMed Central

Osteen, K G; Rodgers, W H; Gaire, M; Hargrove, J T; Gorstein, F; Matrisian, L M

1994-01-01

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase. Images PMID:7937850
Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition.

PubMed

Ghule, Prachi N; Seward, David J; Fritz, Andrew J; Boyd, Joseph R; van Wijnen, Andre J; Lian, Jane B; Stein, Janet L; Stein, Gary S

2018-05-10

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3-dimensional context of nuclear organization is reflected by two well documented observations. DNA replication-dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone-related requirements for G1/S-phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci. © 2018 Wiley Periodicals, Inc.
Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

PubMed Central

Peyton, Kelly J.; Shebib, Ahmad R.; Azam, Mohammad A.; Liu, Xiao-ming; Tulis, David A.; Durante, William

2012-01-01

Bilirubin is a heme metabolite generated by the concerted action of the enzymes heme oxygenase and biliverdin reductase. Although long considered a toxic byproduct of heme catabolism, recent preclinical, and clinical studies indicate the bilirubin exerts beneficial effects in the circulation. In the present study, we determined whether local administration of bilirubin attenuates neointima formation following injury of rat carotid arteries. In addition, the ability of bilirubin to regulate the proliferation and migration of human arterial smooth muscle cells (SMCs) was investigated. Local perivascular administration of bilirubin immediately following balloon injury of rat carotid arteries significantly attenuated neointima formation. Bilirubin-mediated inhibition of neointimal thickening was associated with a significant decrease in ERK activity and cyclin D1 and A protein expression, and an increase in p21 and p53 protein expression in injured blood vessels. Treatment of human aortic SMCs with bilirubin inhibited proliferation and migration in a concentration-dependent manner without affecting cell viability. In addition, bilirubin resulted in a concentration-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and this was paralleled by a decrease in the fraction of cells in the S and G2M phases of the cell cycle. Finally, bilirubin had no effect on mitochondrial function and ATP content of vascular SMCs. In conclusion, these studies demonstrate that bilirubin inhibits neointima formation after arterial injury and this is associated with alterations in the expression of cell cycle regulatory proteins. Furthermore, bilirubin blocks proliferation and migration of human arterial SMCs and arrests SMCs in the G0/G1 phase of the cell cycle. Bilirubin represents an attractive therapeutic agent in treating occlusive vascular disease. PMID:22470341
The cell-cycle interactome: a source of growth regulators?

PubMed

Blomme, Jonas; Inzé, Dirk; Gonzalez, Nathalie

2014-06-01

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

PubMed Central

Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

2014-01-01

Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576
Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R.

PubMed

Li, Ge; Park, Hyeon U; Liang, Dong; Zhao, Richard Y

2010-07-07

Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive. To examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV. These data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.
Circadian rhythms synchronize mitosis in Neurospora crassa.

PubMed

Hong, Christian I; Zámborszky, Judit; Baek, Mokryun; Labiscsak, Laszlo; Ju, Kyungsu; Lee, Hyeyeong; Larrondo, Luis F; Goity, Alejandra; Chong, Hin Siong; Belden, William J; Csikász-Nagy, Attila

2014-01-28

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.
Interferon-α (IFN-α) suppresses HTLV-1 gene expression and cell cycling, while IFN-α combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells

PubMed Central

2013-01-01

Background Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs. Results ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs. Conclusions IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL. PMID:23688327
Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

PubMed Central

Santha, Sreevidya; Bommareddy, Ajay; Rule, Brittny; Guillermo, Ruth; Kaushik, Radhey S.; Young, Alan; Dwivedi, Chandradhar

2013-01-01

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells. PMID:23451128
A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines.

PubMed

Brandi, Giorgio; Paiardini, Mirko; Cervasi, Barbara; Fiorucci, Chiara; Filippone, Paolino; De Marco, Cinzia; Zaffaroni, Nadia; Magnani, Mauro

2003-07-15

Indole-3-carbinol (I3C), autolysis product of glucosinolates present in cruciferous vegetables, has been indicated as a promising agent in preventing the development and progression of breast cancer. I3C has been shown to inhibit the growth of human cancer cells in vitro and possesses anticarcinogenic activity in vivo. Because I3C is unstable and may be converted into many polymeric products in the digestive tract, it is not yet clear whether the biological activity observed can be attributed to I3C or some of its polymeric products. In this study we synthesized a stable I3C cyclic tetrameric derivative and investigated its effects on a panel of human breast cancer cell lines. The I3C tetramer suppressed the growth of both estrogen receptor (ER) -positive (MCF-7, 734B, and BT474) and ER-negative (BT20, MDA-MB-231, and BT539) human breast cancer cell lines, and it was found to induce G(1) cell cycle arrest in a dose-dependent manner without evidence of apoptosis, suggesting a growth arrest via a cytostatic mechanism. At the molecular level, the tetramer inhibited cyclin-dependent kinase (CDK) 6 expression and activity, induced an increase in the level of p27(kip1), and reduced the level of retinoblastoma protein expression. Contrarily to CDK6, the level of CDK4, the other kinase involved in the G(1) phase of the cell cycle, remains unchanged. Interestingly, the tetramer resulted about five times more active than I3C in suppressing the growth of human breast cancer cells. On the whole, our data suggest that the I3C tetrameric derivative is a novel lead inhibitor of breast cancer cell growth that may be a considered a new, promising therapeutic agent for both ER+ and ER- breast cancer.
Modelling the CDK-dependent transcription cycle in fission yeast.

PubMed

Sansó, Miriam; Fisher, Robert P

2013-12-01

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.
Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression†

PubMed Central

Affar, El Bachir; Gay, Frédérique; Shi, Yujiang; Liu, Huifei; Huarte, Maite; Wu, Su; Collins, Tucker; Li, En; Shi, Yang

2006-01-01

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation. PMID:16611997

Formononetin promotes cell cycle arrest via downregulation of Akt/Cyclin D1/CDK4 in human prostate cancer cells.

PubMed

Li, Tianyu; Zhao, Xinge; Mo, Zengnan; Huang, Weihua; Yan, Haibiao; Ling, Zhian; Ye, Yu

2014-01-01

Formononetin is an O-methylated isoflavone isolated from the root of Astragalus membranaceus. It has already been reported that formononetin could inhibit cell proliferation and induce cell apoptosis in several cancers, including prostate cancer. This study aimed to further investigate whether cell cycle arrest is involved in formononetin-mediated antitumor effect in human prostate cancer cells, along with the underlying molecular mechanism. Human prostate cancer cells PC-3 and DU145 were respectively treated with various concentrations of formononetin. The inhibitory effect of formononetin on proliferation of prostate cancer cells was determined using MTT assays and flow cytometry. Next, formononetin-induced alterations in cyclin D1, CDK4 and Akt expression in PC-3 cells were detected by real-time PCR and western blot. Formononetin dose-dependently inhibited prostate cancer cell proliferation via the induction of cell cycle arrest at G0/G1 phase in vitro, which was more evident in PC-3 cells. Meanwhile, concomitant with reduced phosphorylation of Akt in PC-3 cells, formononetin remarkably downregulated expression levels of cyclin D1 and CDK4 in a dose-dependent manner. More interestingly, in the in vivo studies, formononetin showed a noticeable inhibition of tumor growth in recipient mice. Formononetin could exhibit inhibitory activity against human prostate cancer cells in vivo and in vitro, which is associated with G1 cell cycle arrest by inactivation of Akt/cyclin D1/CDK4. Therefore, formononetin may be used as a candidate agent for clinical treatment of prostate cancer in the future.
Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

PubMed

Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4 + T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4 + T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1 + CD4 + T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4 + T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.
Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation

PubMed Central

Celada, Lindsay J.; Rotsinger, Joseph E.; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = −0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression. PMID:27564547
Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Qi-lin; Yang, Tian-lun; Yin, Ji-ye

2009-11-06

Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 {mu}g/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT{sub 1}) mRNA and cyclin E proteinmore » were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 {mu}mol/L) induced HUVECs arrested at G{sub 0}/G{sub 1}, enhanced the expression level of AT{sub 1} mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT{sub 1} mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G{sub 0}/G{sub 1} and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon

Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Resmore » mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI3K, AKT, and NFκB protein expressions in tumor and xenografts. • Res-induced-p21 inhibited DNA repair by modulating Fen-1 binding to PCNA complex.« less
Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

PubMed

Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

2012-04-15

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division. Published by Elsevier GmbH.
The molecular basis of cytotoxicity of α-spinasterol from Ganoderma resinaceum: Induction of apoptosis and overexpression of p53 in breast and ovarian cancer cell lines.

PubMed

Sedky, Nada K; El Gammal, Zaynab H; Wahba, Amir E; Mosad, Eman; Waly, Zahraa Y; El-Fallal, Amira Ali; Arafa, Reem K; El-Badri, Nagwa

2018-05-01

Despite advances in therapy of breast and ovarian cancers, they still remain among the most imperative causes of cancer death in women. The first can be considered one of the most widespread diseases among females, while the latter is more lethal and needs prompt treatment. Thus, the research field can still benefit from discovery of new compounds that can be of potential use in management of these grave illnesses. We hereby aimed to assess the antitumor activity of the phytosterol α-spinasterol isolated from Ganoderma resinaceum mushroom on human breast cancer cell lines (MCF-7, MDA-MB-231), as well as, on human ovarian cancer cell line (SKOV-3). The anti-tumor activity of α-spinasterol, isolated from the mycelial extract of the Egyptian G. resinaceum, on human breast and ovarian cancer cell lines was evaluated by MTT cell viability assay and AnnexinV/propidium iodide apoptosis assay. The molecular mechanism underlying this effect was assessed by the relative expression of the following markers; tumor suppressor (p53, BRCA1, BRCA2), apoptotic marker (Bax) and cell cycle progression markers (cyclin dependent kinases cdk4/6) using real-time PCR. Cell cycle analysis was performed for the three investigated cancer cell lines to explore the effect on cell cycle progression. Our findings showed that α-spinasterol exhibited a higher antitumor activity on MCF-7 cells relative to SKOV-3 cells, while its lowest antitumor activity was against MDA-MB-231 cells. A significant increase in the expression of p53 and Bax was observed in cells treated with α-spinasterol, while cdk4/6 were significantly down-regulated upon exposure to α-spinasterol. Cell cycle analysis of α-spinasterol treated cells showed a G 0 -G 1 arrest. In conclusion, α-spinasterol isolated from G. resinaceum mushroom exerts a potent inhibitory activity on breast and ovarian cancer cell lines in a time- and dose-dependent manner. This can be reasonified in lights of the compound's ability to increase p53 and Bax expressions, and to lower the expression of cdk4/6. © 2017 Wiley Periodicals, Inc.
Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line

PubMed Central

Tesfazghi, Sara; Eide, Jacob; Dammalapati, Ajitha; Korlesky, Colin; Wyche, Thomas P; Bugni, Tim S; Chen, Herbert; Jaskula-Sztul, Renata

2013-01-01

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC. Thiocoraline is a potent Notch pathway activator and an inhibitor of medullary thyroid cancer cell line (MTC-TT) cell proliferation at low nanomolar concentrations. These results provide evidence for the use of thiocoraline as a potential treatment for intractable MTC. PMID:24403239
Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

PubMed

Bruzauskaite, Ieva; Bironaite, Daiva; Bagdonas, Edvardas; Skeberdis, Vytenis Arvydas; Denkovskij, Jaroslav; Tamulevicius, Tomas; Uvarovas, Valentinas; Bernotiene, Eiva

2016-04-30

The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs. hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone. Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores. mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.
Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.
Cortactin modulates RhoA activation and expression of Cip/Kip cyclin-dependent kinase inhibitors to promote cell cycle progression in 11q13-amplified head and neck squamous cell carcinoma cells.

PubMed

Croucher, David R; Rickwood, Danny; Tactacan, Carole M; Musgrove, Elizabeth A; Daly, Roger J

2010-11-01

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) and inhibition of S-phase entry. These effects were associated with increased binding of p21(WAF1/Cip1) and p27(Kip1) to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21(WAF1/Cip1) and p27(Kip1) at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27(Kip1) and p57(Kip2) for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
Andrographolide Induces Cell Cycle Arrest and Apoptosis of Chondrosarcoma by Targeting TCF-1/SOX9 Axis.

PubMed

Zhang, Huan-Tian; Yang, Jie; Liang, Gui-Hong; Gao, Xue-Juan; Sang, Yuan; Gui, Tao; Liang, Zu-Jian; Tam, Man-Seng; Zha, Zhen-Gang

2017-12-01

Chondrosarcoma is the second most malignant bone tumor with poor prognosis and limited treatment options. Thus, development of more effective treatments has become urgent. Recently, natural compounds derived from medicinal plants have emerged as promising therapeutic options via targeting multiple key cellular molecules. Andrographolide (Andro) is such a compound, which has previously been shown to induce cell cycle arrest and apoptosis in several human cancers. However, the molecular mechanism through which Andro exerts its anti-cancer effect on chondrosarcoma remains to be elucidated. In the present study, we showed that Andro-induced G2/M cell cycle arrest of chondrosarcoma by fine-tuning the expressions of several cell cycle regulators such as p21, p27, and Cyclins, and that prolonged treatment of cells with Andro caused pronounced cell apoptosis. Remarkably, we found that SOX9 was highly expressed in poor-differentiated chondrosarcoma, and that knockdown of SOX9 suppressed chondrosarcoma cell growth. Further, our results showed that Andro dose-dependently down-regulated SOX9 expression in chondrosarcoma cells. Concomitantly, an inhibition of T cell factor 1 (TCF-1) mRNA expression and an enhancement of TCF-1 protein degradation by Andro were observed. In contrast, the expression and subcellular localization of β-catenin were not altered upon the treatment of Andro, suggesting that β-catenin might not function as the primary target of Andro. Additionally, we provided evidence that there was a mutual regulation between TCF-1 and SOX9 in chondrosarcoma cells. In conclusion, these results highlight the potential therapeutic effects of Andro in treatment of chondrosarcoma via targeting the TCF-1/SOX9 axis. J. Cell. Biochem. 118: 4575-4586, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
The influence of high glucose on the Cip/Kip family expression profiles in HRECs.

PubMed

Tian, Jingyi; Ma, Hongjie; Luo, Yan; Hu, Andina; Lin, Shaofen; Li, Tao; Guo, Kai; Li, Jing; Cai, Meng; Tang, Shibo

2013-12-01

Neovascularization is the main characteristic of the proliferative stage of diabetic retinopathy. It has been proven that cell cycle regulation is involved in angiogenesis. The cell cycle regulators, Cip/Kip protein family, belong to the cyclin-dependent kinase inhibitors, are versatile proteins, and except for their function in cell cycle regulation, they also participate in transcription, apoptosis and migration. The expression profiles of the Cip/Kip family in human retina microvascular endothelial cells (HRECs) under normal or high glucose conditions has not been described before. This study was undertaken to determine the expression profiles of the Cip/Kip family proteins, e.g., proteins which are influenced by high glucose and in what manner. Western blot and immunofluorescence analyses were used to investigate the protein expression profiles. Only p21(cip1) and p27(kip1) were detected in HRECs, and they were located in the nucleus. P21(cip1) protein abundance was higher than p27(kip1) in HRECs. Incubation of HRECs in medium containing 30 mM D-glucose for 48 h resulted in downregulation of p21(cip1) protein expression, but had no influence on p27(kip1) protein levels or p21(cip1) mRNA abundance. These results were accompanied by cell cycle G1 phase exit and a lower cell survival rate. Our data show for the first time that high glucose changes the Cip/Kip family expression profiles in HRECs, which may be the foundation for the investigation of the role of the Cip/Kip family in the pathogenesis of diabetic retinopathy.
The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

PubMed

Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

2010-06-01

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.
Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle.

PubMed

Turkekul, Kader; Colpan, R Dilsu; Baykul, Talha; Ozdemir, Mehmet D; Erdogan, Suat

2018-03-01

Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c , p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed.
Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle

PubMed Central

Turkekul, Kader; Colpan, R. Dilsu; Baykul, Talha; Ozdemir, Mehmet D.

2018-01-01

Background Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Methods Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Results Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c, p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Conclusions Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed. PMID:29629344
Jasmonate Controls Leaf Growth by Repressing Cell Proliferation and the Onset of Endoreduplication while Maintaining a Potential Stand-By Mode1[W][OA

PubMed Central

Noir, Sandra; Bömer, Moritz; Takahashi, Naoki; Ishida, Takashi; Tsui, Tjir-Li; Balbi, Virginia; Shanahan, Hugh; Sugimoto, Keiko; Devoto, Alessandra

2013-01-01

Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coi1-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1-dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular “stand-by mode” by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1-dependent leaf growth inhibition. PMID:23439917
Experimental study on inhibitory effects of histone deacetylase inhibitor MS-275 and TSA on bladder cancer cells.

PubMed

Qu, Wei; Kang, Yin-Dong; Zhou, Mei-Sheng; Fu, Li-Li; Hua, Zhen-Hao; Wang, Li-Ming

2010-01-01

To investigate the inhibitory effect of histone deacetylase (HDAC) inhibitors (MS-275 and TSA) on T24 human bladder cancer cells in vitro, and explore the possible mechanism. The MTT assay was employed to evaluate the inhibitory effect of MS-275 and TSA on T24 cell growth. FCM was used to analyze the variation of T24 cell cycle distribution and the apoptotic ratio after T24 cells were treated with MS-275 and TSA. Histone acetylation level was detected by Western blot. mRNA expression of p21 WAF1/CIP1, cyclin A, and cyclin E was measured by FQ-PCR. Dynamic changes of Bcl-2 and bax expression were detected by FCM. MS-275 and TSA inhibited T24 cell growth in a concentration and time-dependent manner. Treatment with 4 μmol/l MS-275 or 0.4 μmol/l TSA blocked cell cycling in the G0/G1 phase and induced a significant increase in cell apoptosis. MS-275 and TSA significantly increased the level of histone acetylation, induced p21CIP1WAF1 mRNA expression, and inhibited cyclin A mRNA expression, though no significant effect was observed on cyclin E. Bcl-2 expression was down-regulated, while bax expression was up-regulated. HDAC inhibitors can block bladder cancer cell cycle in vitro and induce apoptosis. The molecular mechanism may be associated with increased level of histone acetylation, down-regulation of p21WAF1/CIP1 expression, up-regulation of cyclin A expression, and dynamic change of bcl-2 and bax expression. Copyright © 2010 Elsevier Inc. All rights reserved.
Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro.

PubMed

Zhu, Lei; Shan, Lu Juan; Liu, Yue Jian; Chen, Dan; Xiao, Xiao Guang; Li, Yan

2014-12-01

Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells. The induction of HCC cell apoptosis by UDCA was dose-dependent and time-dependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.
Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

PubMed

Zhang, X; Xu, Q; Saiki, I

2000-01-01

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

A novel Fizzy/Cdc20-dependent mechanism suppresses necrosis in neural stem cells

PubMed Central

Kuang, Chaoyuan; Golden, Krista L.; Simon, Claudio R.; Damrath, John; Buttitta, Laura; Gamble, Caitlin E.; Lee, Cheng-Yu

2014-01-01

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis. PMID:24598157
Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

PubMed

Liu, Chenglin; Cui, Peng; Huang, Tao

2017-01-01

The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells.

PubMed

Nagahashi, Kotomi; Umemura, Kazuo; Kanayama, Naohiro; Iwaki, Takayuki

2017-04-01

Mammalian gamma-glutamyl carboxylase and reduced vitamin K are indispensable for synthesis of mature mammalian vitamin K dependent proteins including some of blood coagulation factors (factors II, VII, IX, and X). It was well known that Drosophila melanogaster expressed gamma-glutamyl carboxylase and possessed a vit.K cycle although native substrates for them have not been identified yet. Despite the potential capability of gamma carboxylation in D. melanogaster derived cells such as S2 cells, Drosophila gamma-glutamyl carboxylase failed to gamma carboxylate a peptide fused to the human coagulation factor IX propeptide. Thus, it had been believed that the Drosophila system was not adequate to synthesize mammalian vit.K dependent proteins. Indeed, we previously attempted to synthesize biologically active factor VII in S2 cells although we were not able to obtain it. However, recently, a successful transient expression of biologically active human factor IX from S2 cells was reported. In the present study, several expression vectors which enable expressing mammalian GGCX, VKORC1, and/or PDIA2 along with F7 were developed. S2 cells transfected with pMKA85, pMAK86, and pMAK219 successfully synthesized active FVII. Thus, mammalian GGCX was indispensable to synthesize active FVII while mammalian VKORC1 and PDIA2 were not critical but supportive factors for S2 cells.
Tea polyphenols induce S phase arrest and apoptosis in gallbladder cancer cells

PubMed Central

Wang, Jiaqi; Pan, Yixuan; Hu, Jiacheng; Ma, Qiang; Xu, Yi; Zhang, Yijian; Zhang, Fei; Liu, Yingbin

2018-01-01

Gallbladder cancer (GBC) is the most common malignancy in the biliary tract. Without effective treatment, its prognosis is notoriously poor. Tea polyphenols (TPs) have many pharmacological and health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-thrombotic, antibacterial, and vasodilatory properties. However, the anti-cancer effect of TPs in human gallbladder cancer has not yet been determined. Cell viability and colony formation assay were used to investigate the cell growth. Cell cycle and apoptosis were evaluated by flow cytometry analysis. Western blot assay was used to detect the expression of proteins related to cell cycle and apoptosis. Human tumor xenografts were used to examine the effect of TPs on gallbladder cancer cells in vivo. TPs significantly inhibited cell growth of gallbladder cancer cell lines in a dose- and time-dependent manner. Cell cycle progression in GBC cells was blocked at the S phase by TPs. TPs also induced mitochondrial-related apoptosis in GBC cells by upregulating Bax, cleaved caspase-3, and cleaved PARP expressions and downregulating Bcl-2, cyclin A, and Cdk2 expressions. The effects of TPs on GBC were further proven in vivo in a mouse xenograft model. Our study is the first to report that TPs inhibit GBC cell growth and these compounds may have potential as novel therapeutic agents for treating gallbladder cancer. PMID:29513793
Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

PubMed Central

Ligueros, M.; Jeoung, D.; Tang, B.; Hochhauser, D.; Reidenberg, M. M.; Sonenberg, M.

1997-01-01

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9218727
Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control

PubMed Central

Chávez, Santiago; Eastman, Guillermo; Smircich, Pablo; Becco, Lorena Lourdes; Oliveira-Rizzo, Carolina; Fort, Rafael; Potenza, Mariana; Garat, Beatriz; Sotelo-Silveira, José Roberto

2017-01-01

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control. PMID:29182646
Life in the Clouds of Venus? An Experimental Synthetic Biology Approach

NASA Technical Reports Server (NTRS)

Rothschild, L. J.; Paulino-Lima, I. G.; Amatya, D.; Bajar, B.; Geilich, B.; Hu, J.; Jackson, C. J.

2015-01-01

The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.
Induction of cell cycle changes and modulation of apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression by water extracts of I'm-Yunity™ (PSP)

PubMed Central

Hsieh, Tze-chen; Wu, Peili; Park, Spencer; Wu, Joseph M

2006-01-01

Background I'm-Yunity™ (PSP) is a mushroom extract derived from deep-layer cultivated mycelia of the patented Cov-1 strain of Coriolus versicolor (CV), which contains as its main bioactive ingredient a family of polysaccharo-peptide with heterogeneous charge properties and molecular sizes. I'm-Yunity™ (PSP) is used as a dietary supplement by cancer patients and by individuals diagnosed with various chronic diseases. Laboratory studies have shown that I'm-Yunity™ (PSP) enhances immune functions and also modulates cellular responses to external challenges. Recently, I'm-Yunity™ (PSP) was also reported to exert potent anti-tumorigenic effects, evident by suppression of cell proliferation and induction of apoptosis in malignant cells. We investigate the mechanisms by which I'm-Yunity™ (PSP) elicits these effects. Methods Human leukemia HL-60 and U-937 cells were incubated with increasing doses of aqueous extracts of I'm-Yunity™ (PSP). Control and treated cells were harvested at various times and analyzed for changes in: (1) cell proliferation and viability, (2) cell cycle phase transition, (3) induction of apoptosis, (4) expression of cell cycle, apoptogenic/anti-apoptotic, and extracellular regulatory proteins. Results Aqueous extracts of I'm-Yunity™ (PSP) inhibited cell proliferation and induced apoptosis in HL-60 and U-937 cells, accompanied by a cell type-dependent disruption of the G1/S and G2/M phases of cell cycle progression. A more pronounced growth suppression was observed in treated HL-60 cells, which was correlated with time- and dose-dependent down regulation of the retinoblastoma protein Rb, diminution in the expression of anti-apoptotic proteins bcl-2 and survivin, increase in apoptogenic proteins bax and cytochrome c, and cleavage of poly(ADP-ribose) polymerase (PARP) from its native 112-kDa form to the 89-kDa truncated product. Moreover, I'm-Yunity™ (PSP)-treated HL-60 cells also showed a substantial decrease in p65 and to a lesser degree p50 forms of transcription factor NF-κB, which was accompanied by a reduction in the expression of cyclooxygenase 2 (COX2). I'm-Yunity™ (PSP) also elicited an increase in STAT1 (signal transducer and activator of transcription) and correspondingly, decrease in the expression of activated form of ERK (extracellular signal-regulated kinase). Conclusion Aqueous extracts of I'm-Yunity™ (PSP) induces cell cycle arrest and alterations in the expression of apoptogenic/anti-apoptotic and extracellular signaling regulatory proteins in human leukemia cells, the net result being suppression of proliferation and increase in apoptosis. These findings may contribute to the reported clinical and overall health effects of I'm-Yunity™ (PSP). PMID:16965632
Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.
Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

PubMed

Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

2016-10-18

BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.
Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes.

PubMed

Fandy, Tamer E; Jiemjit, Anchalee; Thakar, Manjusha; Rhoden, Paulette; Suarez, Lauren; Gore, Steven D

2014-03-01

Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. 5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC. ©2014 AACR
Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.
Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

PubMed

Kuriyama, Shigeki; Hitomi, Misuzu; Yoshiji, Hitoshi; Nonomura, Takako; Tsujimoto, Tatsuhiro; Mitoro, Akira; Akahane, Takami; Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Masaki, Tsutomu; Uchida, Naohito

2005-08-01

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.
The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Xinming; Shanghai Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031; Gao Xiang

Tob (transducer of ErbB-2) is a negative cell cycle regulator with anti-proliferative activity in peripheral tissues. Our previous study identified Tob as a protein involved in hippocampus-dependent memory consolidation (M.L. Jin, X.M. Wang, Y.Y. Tu, X.H. Zhang, X. Gao, N. Guo, Z.Q. Xie, G.P. Zhao, N.H. Jing, B.M. Li, Y.Yu, The negative cell cycle regulator, Tob (Transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory, Neuroscience 131 (2005) 647-659). Here, we provide evidence that Tob in the central nervous system is engaged in acquisition of motor skill. Tob has a relatively high expression in the cerebellum.more » Tob expression is up-regulated in the cerebellum after rats receive training on a rotarod-running task. Rats infused with Tob antisense oligonucleotides into the 4th ventricle exhibit a severe deficit in running on a rotating rod or walking across a horizontally elevated beam.« less
Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

PubMed Central

Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

2013-01-01

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G2/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G2/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression. PMID:23335992
Berberine Induces Cell Cycle Arrest in Cholangiocarcinoma Cell Lines via Inhibition of NF-κB and STAT3 Pathways.

PubMed

Puthdee, Nattapong; Seubwai, Wunchana; Vaeteewoottacharn, Kulthida; Boonmars, Thidarut; Cha'on, Ubon; Phoomak, Chatchai; Wongkham, Sopit

2017-01-01

Berberine is a natural compound found in several herbs. Anticancer activity of berberine was reported in several cancers, however, little is known regarding the effects of berberine against cholangiocarcinoma (CCA). In this study, the growth inhibitory effects of berberine on CCA cell lines and its molecular mechanisms were explored. Cell growth and cell cycle distribution were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The expression levels of cell cycle regulatory proteins were determined by Western blot analysis. Berberine significantly inhibited growth of CCA cell lines in a dose and time dependent fashion. The inhibition was largely attributed to cell cycle arrest at the G1 phase through the reduction of cyclin D1, and cyclin E. Moreover, berberine could reduce the expression and activation of signal transducers and activator of transcription 3 (STAT3) and probably nuclear factor-kappaB (NF-κB) via suppression of extracellular signal-regulated kinase (ERK) 1/2 action. These results highlight the potential of berberine to be a multi-target agent for CCA treatment.
Nickel chloride (NiCl2) in hepatic toxicity: apoptosis, G2/M cell cycle arrest and inflammatory response

PubMed Central

Guo, Hongrui; Cui, Hengmin; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Zhao, Ling; Chen, Kejie; Deng, Jie

2016-01-01

Up to now, the precise mechanism of Ni toxicology is still indistinct. Our aim was to test the apoptosis, cell cycle arrest and inflammatory response mechanism induced by NiCl2 in the liver of broiler chickens. NiCl2 significantly increased hepatic apoptosis. NiCl2 activated mitochondria-mediated apoptotic pathway by decreasing Bcl-2, Bcl-xL, Mcl-1, and increasing Bax, Bak, caspase-3, caspase-9 and PARP mRNA expression. In the Fas-mediated apoptotic pathway, mRNA expression levels of Fas, FasL, caspase-8 were increased. Also, NiCl2 induced ER stress apoptotic pathway by increasing GRP78 and GRP94 mRNA expressions. The ER stress was activated through PERK, IRE1 and ATF6 pathways, which were characterized by increasing eIF2α, ATF4, IRE1, XBP1 and ATF6 mRNA expressions. And, NiCl2 arrested G2/M phase cell cycle by increasing p53, p21 and decreasing cdc2, cyclin B mRNA expressions. Simultaneously, NiCl2 increased TNF-α, IL-1β, IL-6, IL-8 mRNA expressions through NF-κB activation. In conclusion, NiCl2 induces apoptosis through mitochondria, Fas and ER stress-mediated apoptotic pathways and causes cell cycle G2/M phase arrest via p53-dependent pathway and generates inflammatory response by activating NF-κB pathway. PMID:27824316
5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis

PubMed Central

Grace, Marcy B.; Singh, Vijay K.; Rhee, Juong G.; Jackson, William E.; Kao, Tzu-Cheg; Whitnall, Mark H.

2012-01-01

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis. PMID:22843381
5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis.

PubMed

Grace, Marcy B; Singh, Vijay K; Rhee, Juong G; Jackson, William E; Kao, Tzu-Cheg; Whitnall, Mark H

2012-11-01

The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.
Chromatin remodeling and stem cell theory of relativity.

PubMed

Cerny, Jan; Quesenberry, Peter J

2004-10-01

The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.

Molecular identification of PpHDAC1, the first histone deacetylase fron the slime mold Physarum polycephalum.

PubMed

Brandtner, Eva-Maria; Lechner, Thomas; Loidl, Peter; Lusser, Alexandra

2002-01-01

The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression itself.
AZD8055 Exerts Antitumor Effects on Colon Cancer Cells by Inhibiting mTOR and Cell-cycle Progression.

PubMed

Chen, Yun; Lee, Cheng-Hung; Tseng, Bor-Yuan; Tsai, Ya-Hui; Tsai, Huang-Wen; Yao, Chao-Ling; Tseng, Sheng-Hong

2018-03-01

AZD8055 is an inhibitor of mammalian target of rapamycin (mTOR) that can suppress both mTOR complex 1 (mTORC1) and mTORC2. This study investigated the antitumor effects of AZD8055 on colon cancer. The effects of AZD8055 on proliferation, apoptosis, and cell cycle of colon cancer cells, and tumor growth in a mouse colon cancer model were studied. AZD8055 significantly inhibited proliferation and induced apoptosis of colon cancer cells (p<0.05). The phosphorylation of both AKT and S6 kinase 1 (S6K1) was suppressed by AZD8055. AZD8055 also induced G 0 /G 1 cell-cycle arrest, reduced cyclin D1 and increased p27 expression, and suppressed the levels of phospho-cyclin-dependent kinase 2 and phospho-retinoblastoma. Compared to the control, oral administration of AZD8055 significantly suppressed tumor growth in mice (p<0.05). AZD8055 induces cytotoxicity, apoptosis, and cell-cycle arrest of colon cancer cells, and exerts an antitumor effect in mice. It also inhibits the mTOR signaling pathway and mTOR-dependent cell-cycle progression. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

PubMed

Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

PubMed

Matragkou, Christina N; Papachristou, Eleni T; Tezias, Sotirios S; Tsiftsoglou, Asterios S; Choli-Papadopoulou, Theodora; Vizirianakis, Ioannis S

2008-07-01

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL-C14 and MEL-C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G(1)/G(0) phase, and (c) modulate the level of cyclin-dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer-treated MEL cells. 2008 Wiley-Liss, Inc.
The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa->luteal transition.

PubMed

Tong, W; Kiyokawa, H; Soos, T J; Park, M S; Soares, V C; Manova, K; Pollard, J W; Koff, A

1998-09-01

The involvement of cyclin-dependent kinase inhibitors in differentiation remains unclear: are the roles of cyclin-dependent kinase inhibitors restricted to cell cycle arrest; or also required for completion of the differentiation program; or both? Here, we report that differentiation of luteal cells can be uncoupled from growth arrest in p27-deficient mice. In these mice, female-specific infertility correlates with a failure of embryos to implant at embryonic day 4.5. We show by ovarian transplant and hormone reconstitution experiments that failure to regulate luteal cell estradiol is one physiological mechanism for infertility in these mice. This failure is not due to a failure of p27-deficient granulosa cells to differentiate after hormonal stimulation; P450scc, a marker for luteal progesterone biosynthesis, is expressed and granulosa cell-specific cyclin D2 expression is reduced. However, unlike their wild-type counterparts, p27-deficient luteal cells continue to proliferate for up to 3.5 days after hormonal stimulation. By day 5.5, however, these cells withdraw from the cell cycle, suggesting that p27 plays a role in the early events regulating withdrawal of cells from the cell cycle. We have further shown that in the absence of this timely withdrawal, estradiol regulation is perturbed, explaining in part how fertility is compromised at the level of implantation. These data support the interpretation of our previous observations on oligodendrocyte differentiation about a role for p27 in establishing the nonproliferative state, which in some cases (oligodendrocytes) is required for differentiation, whereas in other cases it is required for the proper functioning of a differentiated cell (luteal cell).
[Effects of sinensetin on proliferation and apoptosis of human gastric cancer AGS cells].

PubMed

Dong, Yang; Ji, Guang; Cao, Aili; Shi, Jianrong; Shi, Hailian; Xie, Jianqun; Wu, Dazheng

2011-03-01

To study the effects and mechanisms of sinensetin on proliferation and apoptosis of human AGS gastric cancer cells. MTT assay was used to detect the growth inhibition rates of human AGS gastric cancer cells treated with sinsesectin in different concentrations and times. The cell cycle distribution was measured by flow cytometry. The apoptosis was examined by Annexin-FITC/PI staining and DNA fragment analysis. The apoptosis morphology was observed by inverted fluorescence microscope after Hoechst 33342 staining. The protein expressions of p21 and p53 were detected by western blot. MTT assay showed that sinensetin inhibited the growth of AGS gastric cancer cells in a dose- and time-dependent manner. Sinensetin blocked AGS cells in G2/ M and increased the apoptosis rates of AGS cells in a dose-dependent manner. DNA ladder was observed in cells treated with 60 micromol x L(-1) sinensetin for 48 h. The typical apoptotic morphological changes including cell nucleus shrinkage, chromatin condensation and apoptotic bodies were observed when treated with different dose of sinensetin. Western blot showed that sinensetin increased expressions of p53 and p21 in a dose-dependent manner. Sinensetin could inhibit human AGS gastric cancer cells proliferation and induce cell cycle block in G2/M phase and apoptosis. The up regulation of p53 and p21 protein might be one of the mechanisms.
Magnolol attenuates neointima formation by inducing cell cycle arrest via inhibition of ERK1/2 and NF-kappaB activation in vascular smooth muscle cells.

PubMed

Karki, Rajendra; Ho, Oak-Min; Kim, Dong-Wook

2013-03-01

Endovascular injury induces switching of contractile phenotype of vascular smooth muscle cells (VSMCs) to synthetic phenotype, thereby causing proliferation of VSMCs leading to intimal thickening. The purpose of this study was to assess the effect of magnolol on the proliferation of VSMCs in vitro and neointima formation in vivo, as well as the related cell signaling mechanisms. Tumor necrosis factor alpha (TNF-alpha) induced proliferation ofVSMCs was assessed using colorimetric assay. Cell cycle progression and mRNA expression of cell cycle associated molecules were determined by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) respectively. The signaling molecules such as ERK1/2,JNK, P38 and NF-kappaB were determined by Western blot analysis. In addition, rat carotid artery balloon injury model was performed to assess the effect of magnolol on neointima formation in vivo. Oral administration of magnolol significantly inhibited intimal area and intimal/medial ratio (I/M). Our in vitro assays revealed magnolol dose dependently induced cell cycle arrest at G0/G1. Also, magnolol inhibited mRNA and protein expression of cyclin D1, cyclin E, CDK4 and CDK2 in vitro and in vivo. The cell cycle arrest was associated with inhibition of ERK1/2 phosphorylation and NF-kappaB translocation. Magnolol suppressed proliferation of VSMCs in vitro and attenuated neointima formation in vivo by inducing cell cycle arrest at G0/G1 through modulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Thus, the results suggest that magnolol could be a potential therapeutic candidate for the prevention of restenosis and atherosclerosis.
Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

PubMed

Gao, Lin-Lin; Feng, Lei; Yao, Shu-Tong; Jiao, Peng; Qin, Shu-Cun; Zhang, Wei; Zhang, Ya-Bin; Li, Fu-Rong

2011-01-01

Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.
Antiproliferative action of Xylopia aethiopica fruit extract on human cervical cancer cells.

PubMed

Adaramoye, Oluwatosin A; Sarkar, Jayanta; Singh, Neetu; Meena, Sanjeev; Changkija, Bendangla; Yadav, Prem P; Kanojiya, Sanjeev; Sinha, Sudhir

2011-10-01

The anticancer potential of Xylopia aethiopica fruit extract (XAFE), and the mechanism of cell death it elicits, was investigated in various cell lines. Treatment with XAFE led to a dose-dependent growth inhibition in most cell lines, with selective cytotoxicity towards cancer cells and particularly the human cervical cancer cell line C-33A. In this study, apoptosis was confirmed by nuclear fragmentation and sub-G(0)/G(1) phase accumulation. The cell cycle was arrested at the G(2)/M phase with a decreased G(0)/G(1) population. A semi-quantitative gene expression study revealed dose-dependent up-regulation of p53 and p21 genes, and an increase in the Bax/Bcl-2 ratio. These results indicate that XAFE could be a potential therapeutic agent against cancer since it inhibits cell proliferation, and induces apoptosis and cell cycle arrest in C-33A cells. Copyright © 2011 John Wiley & Sons, Ltd.
Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.
Antiviral effects of artesunate on polyomavirus BK replication in primary human kidney cells.

PubMed

Sharma, Biswa Nath; Marschall, Manfred; Henriksen, Stian; Rinaldo, Christine Hanssen

2014-01-01

Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 μM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 μM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 μM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2'-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 μM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions.
Antiviral Effects of Artesunate on Polyomavirus BK Replication in Primary Human Kidney Cells

PubMed Central

Sharma, Biswa Nath; Marschall, Manfred; Henriksen, Stian

2014-01-01

Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 μM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 μM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 μM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2′-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 μM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions. PMID:24145549
Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

PubMed Central

Róna, Gergely; Borsos, Máté; Ellis, Jonathan J; Mehdi, Ahmed M; Christie, Mary; Környei, Zsuzsanna; Neubrandt, Máté; Tóth, Judit; Bozóky, Zoltán; Buday, László; Madarász, Emília; Bodén, Mikael; Kobe, Bostjan; Vértessy, Beáta G

2014-01-01

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle. PMID:25483092
Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853
Preclinical evaluation of transcriptional targeting strategy for human hepatocellular carcinoma in an orthotopic xenograft mouse model.

PubMed

Sia, Kian Chuan; Huynh, Hung; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Hui, Kam Man; Lam, Paula Yeng Po

2013-08-01

Gene regulation of many key cell-cycle players in S-, G(2) phase, and mitosis results from transcriptional repression in their respective promoter regions during the G(0) and G(1) phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle-dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma.
The Expression of Glyceraldehyde-3-Phosphate Dehydrogenase Associated Cell Cycle (GACC) Genes Correlates with Cancer Stage and Poor Survival in Patients with Solid Tumors

PubMed Central

Wang, Dunrui; Moothart, Daniel R.; Lowy, Douglas R.; Qian, Xiaolan

2013-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis. PMID:23620736
[Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension].

PubMed

Rylova, Iu V; Buravkova, L B

2013-01-01

We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.
Flow cytometric monitoring of hormone receptor expression in human solid tumors

NASA Astrophysics Data System (ADS)

Krishan, Awtar

2002-05-01

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.
Genistein induced anticancer effects on pancreatic cancer cell lines involves mitochondrial apoptosis, G0/G1cell cycle arrest and regulation of STAT3 signalling pathway.

PubMed

Bi, Yi-Liang; Min, Min; Shen, Wei; Liu, Yan

2018-01-15

Genistein is a natural flavonoid that has been reported to exhibit anticancer effects against different types of cancers which include, but are not limited to, breast and oral squamous cell carcinoma. The present study was designed to evaluate the anticancer effects of the natural flavonoid genistein against pancreatic cancer cell lines and to explore the underlying mechanism. Antiproliferative activity was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining. DNA damage was assessed by comet assay. Reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. Cell migration was examined by wound healing assay. Protien expressions were determined by western blotting. Antiproliferative assay revealed that genistein reduced the cell viability of pancreatic cancer cells in a dose dependent manner with an IC 50 of 20 and 25 µM against Mia-PaCa2 and PANC-1 cancer cell lines respectively. However, its antiproliferative effects were less pronounced against non-cancerous pancreatic ductal epithelial cell line (H6C7) as evident from the IC 50 of 120 µM. Genistein induced significant morphological changes in pancreatic cancer cells and triggered cell cycle arrest in G 0 /G 1 phase. DAPI staining and flow cytometric analysis revealed that genistein induced apoptosis in a dose dependent manner through generation of substantial amounts of ROS and reduction of MMP. However, treatment of the pancreatic cancer with genistein and ascorbic acid could abrogate the effects of genistein on cell viability. Protien expression analysis revealed that genistein upregulated cytosolic cytochrome c, Bax, cleaved Caspase-3 and cleaved caspase-9 expressions with concomitant downregulation of Bcl-2 expression. Moreover, genistein inhibited the phosphorylation of signal transducer and activator of transcription STAT3 proteins and downregulated the expression of survivin, cyclin D1 and ALDH1A1 in Mia-PaCa2 cells in a dose dependent manner. Interestingly, genistein could inhibit the cell migration potential of the Mia-PaCa2 cells which was further associated with the downregulation of metalloproteinases (MPP-2 and MPP-9). Taken together, we propose that genistein exerts anticancer activity in pancreatic cancer cells through induction of ROS mediated mitochondrial apoptosis, cell cycle arrest and regulation of STAT3 and may therefore prove beneficial in the management of pancreatic cancers cancer. Copyright © 2017 Elsevier GmbH. All rights reserved.
An R132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein in Glioma Cells

PubMed Central

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1R132H-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1R132H-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progressionof the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation. PMID:24077277

An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.

PubMed

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.
A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

PubMed Central

Millard, S. Sean; Vidal, Anxo; Markus, Maurice; Koff, Andrew

2000-01-01

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals. PMID:10913178
FOXC2 regulates the G2/M transition of stem cell-rich breast cancer cells and sensitizes them to PLK1 inhibition

PubMed Central

Pietilä, Mika; Vijay, Geraldine V.; Soundararajan, Rama; Yu, Xian; Symmans, William F.; Sphyris, Nathalie; Mani, Sendurai A.

2016-01-01

Cancer cells with stem cell properties (CSCs) underpin the chemotherapy resistance and high therapeutic failure of triple-negative breast cancers (TNBCs). Even though CSCs are known to proliferate more slowly, they are sensitive to inhibitors of G2/M kinases such as polo-like kinase 1 (PLK1). Understanding the cell cycle regulatory mechanisms of CSCs will help target these cells more efficiently. Herein, we identify a novel role for the transcription factor FOXC2, which is mostly expressed in CSCs, in the regulation of cell cycle of CSC-enriched breast cancer cells. We demonstrate that FOXC2 expression is regulated in a cell cycle-dependent manner, with FOXC2 protein levels accumulating in G2, and rapidly decreasing during mitosis. Knockdown of FOXC2 in CSC-enriched TNBC cells delays mitotic entry without significantly affecting the overall proliferation rate of these cells. Moreover, PLK1 activity is important for FOXC2 protein stability, since PLK1 inhibition reduces FOXC2 protein levels. Indeed, FOXC2 expressing CSC-enriched TNBC cells are sensitive to PLK1 inhibition. Collectively, our findings demonstrate a novel role for FOXC2 as a regulator of the G2/M transition and elucidate the reason for the observed sensitivity of CSC-enriched breast cancer cells to PLK1 inhibitor. PMID:27064522
Xanthorrhizol, a natural sesquiterpenoid, induces apoptosis and growth arrest in HCT116 human colon cancer cells.

PubMed

Kang, You-Jin; Park, Kwang-Kyun; Chung, Won-Yoon; Hwang, Jae-Kwan; Lee, Sang Kook

2009-11-01

Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. In our previous studies, xanthorrhizol suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, inhibited cancer cell growth, and exerted an anti-metastatic effect in an animal model. However, the exact mechanisms for its inhibitory effects against cancer cell growth have not yet been fully elucidated. In the present study, we investigated the growth inhibitory effect of xanthorrhizol on cancer cells. Xanthorrhizol dose-dependently exerted antiproliferative effects against HCT116 human colon cancer cells. Xanthorrhizol also arrested cell cycle progression in the G0/G1 and G2/M phase and induced the increase of sub-G1 peaks. Cell cycle arrest was highly correlated with the downregulation of cyclin A, cyclin B1, and cyclin D1; cyclin-dependent kinase 1 (CDK1), CDK2, and CDK4; proliferating cell nuclear antigen; and inductions of p21 and p27, cyclin-dependent kinase inhibitors. The apoptosis by xanthorrhizol was markedly evidenced by induction of DNA fragmentation, release of cytochrome c, activation of caspases, and cleavage of poly-(ADP-ribose) polymerase. In addition, xanthorrhizol increased the expression and promoter activity of pro-apoptotic non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). These findings provide one plausible mechanism for the growth inhibitory activity of xanthorrhizol against cancer cells.
Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

PubMed

Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

2014-05-15

The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.
Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models.

PubMed

Lehmann, Christian; Friess, Thomas; Birzele, Fabian; Kiialainen, Anna; Dangl, Markus

2016-06-28

Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.
Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells

PubMed Central

Choi, Y; Lim, SY; Jeong, HS; Koo, KA; Sung, SH; Kim, YC

2009-01-01

Background and purpose: We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45α, gadd45γ and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA. PMID:19466985
The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins.

PubMed

Bhattacharya, Abhishek; Li, Ke; Quiquand, Manon; Rimesso, Gerard; Baker, Nicholas E

2017-11-15

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets. Copyright © 2017 Elsevier Inc. All rights reserved.
A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

PubMed

Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike; Buchrieser, Carmen

2017-01-10

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated. Copyright © 2017 Oliva et al.
Sulforaphane Increases Cyclin-Dependent Kinase Inhibitor, p21 Protein in Human Oral Carcinoma Cells and Nude Mouse Animal Model to Induce G2/M Cell Cycle Arrest

PubMed Central

Kim, Jun-Hee; Han Kwon, Ki; Jung, Ji-Youn; Han, Hye-Suk; Hyun Shim, Jung; Oh, SeJun; Choi, Kyeong-Hee; Choi, Eun-Sun; Shin, Ji-Ae; Leem, Dae-Ho; Soh, Yunjo; Cho, Nam-Pyo; Cho, Sung-Dae

2010-01-01

Previously, our group reported that sulforaphane (SFN), a naturally occurring chemopreventive agent from cruciferous vegetables, effectively inhibits the proliferation of KB and YD-10B human oral squamous carcinoma cells by causing apoptosis. In this study, treatment of 20 and 40 µM of SFN for 12 h caused a cell cycle arrest in the G2/M phase. Cell cycle arrest induced by SFN was associated with a significant increase in the p21 protein level and a decrease in cyclin B expression, but there was no change in the cyclin A protein level. In addition, SFN increased the p21 promoter activity significantly. Furthermore, SFN induced p21 protein expression in a nude mouse xenograft model suggesting that SFN is a potent inducer of the p21 protein in human oral squamous carcinoma cells. These findings show that SFN is a promising candidate for molecular-targeting chemotherapy against human oral squamous cell carcinoma. PMID:20104266
Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells.

PubMed

Canales, Jimena; Valenzuela, Manuel; Bravo, Jimena; Cerda-Opazo, Paulina; Jorquera, Carla; Toledo, Héctor; Bravo, Denisse; Quest, Andrew F G

2017-01-01

Helicobacter pylori ( H. pylori ) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori -induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.
Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.

PubMed

Fu, Yujie; Kadioglu, Onat; Wiench, Benjamin; Wei, Zuofu; Gao, Chang; Luo, Meng; Gu, Chengbo; Zu, Yuangang; Efferth, Thomas

2015-04-15

The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet. We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis. CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways. CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.
The human HECA interacts with cyclins and CDKs to antagonize Wnt-mediated proliferation and chemoresistance of head and neck cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dowejko, Albert, E-mail: Albert.Dowejko@klinik.uni-regensburg.de; Bauer, Richard; Bauer, Karin

2012-03-10

There is a growing evidence that the human homologue of the Drosophila headcase (HECA) plays an important role in human carcinogenesis. So far specific protein interaction partners and affected signaling pathways of HECA are still elusive. In a recent study we showed that HECA overexpression in oral squamous-cell carcinoma (OSCC) keratinocytes has tumor suppressive effects resulting in a recuperation of cell cycle control concerning the entry and progression of S-phase, G2- and M-phase. Currently, quantitative RT-PCR and immunohistochemical analysis of primary tumor tissue from OSCC patients demonstrate that HECA expression is markedly decreased compared to normal control patients with abundantmore » HECA expression. Additionally, there is nearly no HECA expression in OSCC metastases. Here, we show that HECA expression is negatively controlled by the Wnt-pathway and TCF4, a Wnt related transcription factor, binds to the HECA promoter. Furthermore, immunocytochemistry reveals colocalization of HECA with the cyclin dependent kinase CDK9. Immunoprecipitation experiments and proximity ligation assays further reveal an interaction of HECA with CDK2, CDK9, Cyclin A and Cyclin K, a direct transcriptional target of the p53 tumor suppressor. Silencing HECA in OSCC cell lines leads to a significant increase of cell division and a markedly increased resistance against the chemotherapeutic cisplatin. On the contrary, HECA overexpressing OSCC cell lines show decreased resistance of OSCC cells against cisplatin. Therefore, HECA could be considered as future therapeutic agent against Wnt-dependent tumor progression. -- Highlights: Black-Right-Pointing-Pointer HECA is a new cell cycle regulator with anti-tumor features in head and neck cancer. Black-Right-Pointing-Pointer During tumor progression HECA mRNA and protein expression decrease. Black-Right-Pointing-Pointer The HECA promotor is a direct target of the Wnt/beta-catenin/TCF-pathway. Black-Right-Pointing-Pointer The HECA protein antagonizes Wnt-mediated cell proliferation through interaction with major cell cycle factors. Black-Right-Pointing-Pointer Modulating HECA level confers benefits for engaging tumor cells with cisplatin.« less
Stimulatory effect of icariin on the proliferation of neural stem cells from rat hippocampus.

PubMed

Fu, Xiaolong; Li, Shujun; Zhou, Shaoyu; Wu, Qin; Jin, Feng; Shi, Jingshan

2018-01-29

Icariin (ICA), a major ingredient of Epimediumbrevicornum, has various pharmacological activities including central nervous system protective functions such as the improvement of learning and memory function in mice models of Alzheimer's disease. It has been reported that ICA can promote regeneration of peripheral nerve and functional recovery. The purpose of this study was to investigate the potentiating effect of ICA on the proliferation of rat hippocampal neural stem cells, and explore the possible mechanism involved. Primary neural stem cells were prepared from the hippocampus of newly born SD rats, and cells were cultured in special stem cell culture medium. Neural stem cells were confirmed by immunofluorescence detection of nestin, NSE and GFAP expression. The effect of ICA on the growth and proliferation of the neural stem cells was evaluated by 5-ethynyl-2-deoxyuridine (EdU) labeling of proliferating cells, and photomicrographic images of the cultured neural stem cells. Further, the mechanism of ICA-induced cell proliferation of neural stem cells was investigated by analyzing the gene and protein expression of cell cycle related genes cyclin D1 and p21. The present study showed that icariin promotes the growth and proliferation of neural stem cells from rat hippocampus in a dose-dependent manner. Incubation of cells with icariin resulted in significant increase in the number of stem cell spheres as well as the increased incorporation of EdU when compared with cells exposed to control vehicle. In addition, it was found that icariin-induced effect on neural stem cells is associated with increased mRNA and protein expression of cell cycle genes cyclin D1 and p21. This study evidently demonstrates the potentiating effect of ICA on neural stem cell growth and proliferation, which might be mediated through regulation of cell cycle gene and protein expression promoting cell cycle progression.
Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.

PubMed

Toyota, Yuka; Iwama, Hisakazu; Kato, Kiyohito; Tani, Joji; Katsura, Akiko; Miyata, Miwa; Fujiwara, Shintaro; Fujita, Koji; Sakamoto, Teppei; Fujimori, Takayuki; Okura, Ryoichi; Kobayashi, Kiyoyuki; Tadokoro, Tomoko; Mimura, Shima; Nomura, Takako; Miyoshi, Hisaaki; Morishita, Asahiro; Kamada, Hideki; Yoneyama, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Masaki, Tsutomu

2015-10-01

Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.
Butylated Hydroxyanisole Stimulates Heme Oxygenase-1 Gene Expression and Inhibits Neointima Formation in Rat Arteries

PubMed Central

Liu, Xiao-ming; Azam, Mohammed A.; Peyton, Kelly J.; Ensenat, Diana; Keswani, Amit N.; Wang, Hong; Durante, William

2007-01-01

Objective Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. Methods The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. Results Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in a HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. Conclusions These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease. PMID:17320844
Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

PubMed

de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

2015-10-02

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis. Copyright © 2015. Published by Elsevier Ireland Ltd.
Molecular basis of sodium butyrate-dependent proapoptotic activity in cancer cells.

PubMed

Pajak, B; Orzechowski, A; Gajkowska, B

2007-01-01

This review outlines the molecular events that accompany the antitumor action of sodium butyrate (NaBt). Butyrate, a low-molecular weight four-carbon chain volatile fatty acid (VFA) has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate, that this product of large bowel microbial fermentation of dietary fiber, might evoke the above-mentioned effects by indirect action on genes. NaBt was shown to inhibit histone deacetylase activity, allowing DNA binding of several transcription factors. Higher genomic activity leads to the higher expression of proapoptotic genes, higher level of their protein products and elevated sensitivity to death ligand-induced apoptosis. Cancer cells might be arrested in G1 phase of cell cycle in a p21-dependent manner. Proapoptotic activity of NaBt includes higher expression of membrane death receptors (DR4/5), higher level and activation of Smad3 protein in TGF-beta-dependent apoptotic pathway, lower level of antiapoptotic proteins (cFLIP, XIAP) and activation ofproapoptotic tBid protein. Thus, both intrinsic and extrinsic apoptotic pathways are stimulated to ampify the apoptotic signals. These effects are specific for tumor but not for regular cells. Unique properties of NaBt make this agent a promising metabolic inhibitor to retard tumorigenesis to suppress tumor growth.
Cyclin Dependent Kinase Inhibitors as Targets in Ovarian Cancer

DTIC Science & Technology

2005-10-01

STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The objective of this proposal is to develop gene ...have identified key genes that may be effective targets in ovarian cancer therapy. The first three projects seek to identify alterations in these genes ...that allow for high expression of our key gene (s) in ovarian cancer cells but minimal expression in normal tissues. 15. SUBJECT TERMS Cell cycle control
Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms.

PubMed

Yu, Si; Wang, Lijiao; Cao, Zhixing; Gong, Daoyin; Liang, Qianyi; Chen, Hanting; Fu, Huizhu; Wang, Wenwen; Tang, Xue; Xie, Zihao; He, Yang; Peng, Cheng; Li, Yuzhi

2018-06-01

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon

Voltage-dependent K{sup +} channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G{sub 1}-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletalmore » muscle cell proliferation.« less
Gamma Irradiation Upregulates B-cell Translocation Gene 2 to Attenuate Cell Proliferation of Lung Cancer Cells Through the JNK and NF-κB Pathways.

PubMed

Wang, Peihe; Cai, Yuanyuan; Lin, Dongju; Jiang, Yingxiao

2017-08-07

Gamma ray can promote cancer cell apoptosis and cell cycle arrest. It is often used in the clinical treatment of tumors, including lung cancer. In this study, we aimed to explore the role of gamma ray treatment and its correlation with BTG2 in cell proliferation, apoptosis, and cell cycle arrest regulation in a lung cancer cell line. A549 cell viability, apoptosis rate, and cell cycle were investigated after gamma ray treatment. We then used siRNA for BTG2 to detect the effect of BTG2 knockdown on the progress of gamma ray-treated lung cancer cells. Finally, we investigated the signaling pathway by which gamma ray might regulate BTG2. We found that gamma ray inhibited A549 cell viability and promoted apoptosis and cell cycle arrest, while BTG2 knockdown could relieve the effect caused by gamma ray on A549 cells. Moreover, we confirmed that the effect of BTG2 partly depends on p53 expression and gamma ray-promoting BTG2 expression through the JNK/NF-κB signaling pathway. Our study assessed the possible mechanism of gamma ray in tumor treatment and also investigated the role of BTG2 in gamma ray therapy. All these findings might give a deep understanding of the effect of gamma ray on the progression of lung cancer involving BTG2.
Parathyroid cell growth in patients with advanced secondary hyperparathyroidism: vitamin D receptor and cyclin-dependent kinase inhibitors, p21 and p27.

PubMed

Tokumoto, Masanori; Tsuruya, Kazuhiko; Fukuda, Kyoichi; Kanai, Hidetoshi; Kuroki, Shoji; Hirakata, Hideki; Iida, Mitsuo

2003-06-01

Uraemic patients with advanced secondary hyperparathyroidism (2HPT) have nodular hyperplastic glands with a decreased vitamin D receptor (VDR) density. Previous studies have shown that nodular hyperplasia expressed a significantly lower VDR density as compared with diffuse hyperplasia, and the VDR density negatively correlated with both the glandular weight and the marker of cell proliferation. However, the mechanism by which the decreased VDR density leads to parathyroid cell proliferation remains unclear. In the myelomonocytic cell line, active vitamin D(3) is known to activate the transcription of both p21 and p27, cyclin-dependent kinase inhibitors (CDKIs), regulating the transition from the G(1) to the S phase of the cell cycle, in a VDR-dependent manner. Moreover, the overexpression of p21 and p27 inhibits cell proliferation. In order to elucidate the mechanism of parathyroid cell proliferation, the expression of CDKIs, p21 and p27, and the VDR was analysed immunohistochemically, and compared among nodular and diffuse hyperplastic parathyroid glands, and histologically normal parathyroid glands. The VDR expression in nodular hyperplasias was significantly decreased compared with either diffuse hyperplasias or normal parathyroid glands. The expression of both p21 and p27 was also significantly lower in nodular hyperplasias than in diffuse hyperplasias or normal parathyroid glands. Sections of parathyroid glands with a high expression of nuclear VDR highly expressed both p21 and p27. In nodular hyperplasias, the expression of both p21 and p27 correlated either positively with the nuclear VDR expression or inversely with the glandular weight. Therefore, the reduced expression of p21 and p27, being VDR dependent, is a major pathogenic factor for nodular parathyroid gland growth in advanced 2HPT.
Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

PubMed

Kapoor, Vaishali; Zaharieva, Maya M; Das, Satya N; Berger, Martin R

2012-06-01

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Long non‑coding RNA AK001796 contributes to cisplatin resistance of non‑small cell lung cancer.

PubMed

Liu, Bin; Pan, Chun-Feng; Ma, Teng; Wang, Jun; Yao, Guo-Liang; Wei, Ke; Chen, Yi-Jiang

2017-10-01

Cisplatin (DDP)‑based chemotherapy is the most widely used therapy for non‑small cell lung cancer (NSCLC). However, the existence of chemoresistance has become a major limitation in its efficacy. Long non‑coding RNAs (lncRNAs) have been shown to be involved in chemotherapy drug resistance. The aim of the present study was to investigate the biological role of lncRNA AK001796 in cisplatin‑resistant NSCLC A549/DDP cells. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed to monitor the differences in the expression of AK001796 in cisplatin-resistant (A549/DDP) cells and parental A549 cells. Cellular sensitivity to cisplatin and cell viability were examined using an MTT assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. The expression levels of cell cycle proteins cyclin C (CCNC), baculoviral IAP repeat containing 5 (BIRC5), cyclin‑dependent kinase 1 (CDK1) and G2 and S phase‑expressed 1 (GTSE1) were assessed using RT‑qPCR and western blot analyses. It was found that the expression of AK001796 was increased in A549/DDP cells, compared with that in A549 cells. The knockdown of AK001796 by small interfering RNA reduced cellular cisplatin resistance and cell viability, and resulted in cell‑cycle arrest, with a marked increase in the proportion of A549/DDP cells in the G0/G1 phase. By contrast, the knockdown of AK001796 increased the number of apoptotic cancer cells during cisplatin treatment. It was also shown that the knockdown of AK001796 positively induced the expression of cell apoptosis‑associated factors, CCNC and BIRC5, and suppressed the expression of cell cycle‑associated factors, CDK1 and GTSE5. Taken together, these findings indicated that lncRNA AK001796 increased the resistance of NSCLC cells to cisplatin through regulating cell apoptosis and cell proliferation, and thus provides an attractive therapeutic target for NSCLC.
High expression of Polycomb group protein EZH2 predicts poor survival in salivary gland adenoid cystic carcinoma.

PubMed

Vékony, H; Raaphorst, F M; Otte, A P; van Lohuizen, M; Leemans, C R; van der Waal, I; Bloemena, E

2008-06-01

The prognosis of adenoid cystic carcinoma (ACC), a malignant salivary gland tumour, depends on clinicopathological parameters. To decipher the biological behaviour of ACC, and to identify patients at risk of developing metastases, additional markers are needed. Expression of the cell cycle proteins p53, cyclin D1, p16(INK4a), E2F1 and Ki-67, together with the Polycomb group (PcG) proteins BMI-1, MEL-18, EZH2 and EED was investigated immunohistochemically 21 formalin-fixed, paraffin-embedded primary ACCs in relation to tumour characteristics. ACC revealed significantly increased expression of the cell cycle proteins compared to normal salivary tissue (n = 17). Members of the two PcG complexes displayed mutually exclusive expression in normal salivary gland tissue, with BMI-1 and MEL-18 being abundantly present. In ACC, this expression pattern was disturbed, with EZH2 and EED showing significantly increased expression levels. In univariate analysis, presence of recurrence, poor differentiation and high EZH2 levels (>25% immunopositivity) significantly correlated with unfavourable outcome. ACCs with high proliferative rate (>25% Ki-67 immunopositivity) significantly correlated with high levels of EZH2 and p16. Only the development of recurrence was an independent prognostic factor of survival in multivariate analysis. Expression of PcG complexes and of essential cell cycle proteins is highly deregulated in ACC. Also, EZH2 expression has prognostic relevance in this malignancy.
The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

PubMed

Brodie, Shlomit; Lee, Hae Kyung; Jiang, Wei; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M; Datta, Indrani; Kalkanis, Steve; Ginsberg, Doron; Brodie, Chaya

2017-05-09

Despite advances in novel therapeutic approaches for the treatment of glioblastoma (GBM), the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need to identify molecular mechanisms that play a role in patient survival. Here, we analyzed the expression and functions of a novel lncRNA, TALNEC2 that was identified using RNA seq of E2F1-regulated lncRNAs. TALNEC2 was localized to the cytosol and its expression was E2F1-regulated and cell-cycle dependent. TALNEC2 was highly expressed in GBM with poor prognosis, in GBM specimens derived from short-term survivors and in glioma cells and glioma stem cells (GSCs). Silencing of TALNEC2 inhibited cell proliferation and arrested the cells in the G1\\S phase of the cell cycle in various cancer cell lines. In addition, silencing of TALNEC2 decreased the self-renewal and mesenchymal transformation of GSCs, increased sensitivity of these cells to radiation and prolonged survival of mice bearing GSC-derived xenografts. Using miRNA array analysis, we identified specific miRNAs that were altered in the silenced cells that were associated with cell-cycle progression, proliferation and mesenchymal transformation. Two of the downregulated miRNAs, miR-21 and miR-191, mediated some of TALNEC2 effects on the stemness and mesenchymal transformation of GSCs. In conclusion, we identified a novel E2F1-regulated lncRNA that is highly expressed in GBM and in tumors from patients of short-term survival. The expression of TALNEC2 is associated with the increased tumorigenic potential of GSCs and their resistance to radiation. We conclude that TALNEC2 is an attractive therapeutic target for the treatment of GBM.
The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.
NONO couples the circadian clock to the cell cycle.

PubMed

Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A

2013-01-29

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.
Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620
Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

PubMed Central

Sharma, Chhavi; Vas, Andrea J.; Goala, Payal; Gheewala, Taher M.; Rizvi, Tahir A.

2014-01-01

The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE) alone or in combination with cisplatin by cell viability assay on human breast (MCF-7) and cervical (HeLa) cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index <1). ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2) in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. Thus, neem may be a prospective therapeutic agent to combat gynecological cancers. PMID:24624140
Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

PubMed

Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

2005-11-01

Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in regulation of contact inhibition in these normally non-proliferating cells.
Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

NASA Technical Reports Server (NTRS)

Swarthout, John T.; D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Partridge, Nicola C.

2002-01-01

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.
Phloretin induces cell cycle arrest and apoptosis of human glioblastoma cells through the generation of reactive oxygen species.

PubMed

Liu, Yuanyuan; Fan, Chenghe; Pu, Lv; Wei, Cui; Jin, Haiqiang; Teng, Yuming; Zhao, Mingming; Yu, Albert Cheung Hoi; Jiang, Feng; Shu, Junlong; Li, Fan; Peng, Qing; Kong, Jian; Pan, Bing; Zheng, Lemin; Huang, Yining

2016-06-01

Phloretin, a flavonoid present in various plants, has been reported to exert anticarcinogenic effects. However, the mechanism of its chemo-preventive effect on human glioblastoma cells is not fully understood. This study aimed to investigate the molecular mechanism of phloretin and its associated chemo-preventive effect in human glioblastoma cells. The results indicate that phloretin inhibited cell proliferation by inducing cell cycle arrest at the G0-G1 phase and induced apoptosis of human glioblastoma cells. Phloretin-induced cell cycle arrest was associated with increased expression of p27 and decreased expression of cdk2, cdk4, cdk6, cyclinD and cyclinE. Moreover, the PI3K/AKT/mTOR signaling cascades were suppressed by phloretin in a dose-dependent manner. In addition, phloretin triggered the mitochondrial apoptosis pathway and generated reactive oxygen species (ROS). This was accompanied by the up-regulation of Bax, Bak and c-PARP and the down-regulation of Bcl-2. The antioxidant agents N-acetyl-L-cysteine and glutathione weakened the effect of phloretin on glioblastoma cells. In conclusion, these results demonstrate that phloretin exerts potent chemo-preventive activity in human glioblastoma cells through the generation of ROS.
Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513
Differentiation-associated microRNAs antagonize the Rb–E2F pathway to restrict proliferation

PubMed Central

Marzi, Matteo J.; Puggioni, Eleonora M. R.; Dall'Olio, Valentina; Bucci, Gabriele; Bernard, Loris; Bianchi, Fabrizio; Crescenzi, Marco

2012-01-01

The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation—transcriptional regulation via Rb–E2F and posttranscriptional regulation via miRNAs—confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer. PMID:23027903
Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horiuchi, Shinji; Kato, Kiyoko; Suga, Shin

2005-05-01

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phasemore » of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.« less
Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

PubMed

Oviedo, Pilar J; Sobrino, Agua; Laguna-Fernandez, Andrés; Novella, Susana; Tarín, Juan J; García-Pérez, Miguel-Angel; Sanchís, Juan; Cano, Antonio; Hermenegildo, Carlos

2011-03-30

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.
Development of cell-cycle checkpoint therapy for solid tumors.

PubMed

Tamura, Kenji

2015-12-01

Cellular proliferation is tightly controlled by several cell-cycle checkpoint proteins. In cancer, the genes encoding these proteins are often disrupted and cause unrestrained cancer growth. The proteins are over-expressed in many malignancies; thus, they are potential targets for anti-cancer therapies. These proteins include cyclin-dependent kinase, checkpoint kinase, WEE1 kinase, aurora kinase and polo-like kinase. Cyclin-dependent kinase inhibitors are the most advanced cell-cycle checkpoint therapeutics available. For instance, palbociclib (PD0332991) is a first-in-class, oral, highly selective inhibitor of CDK4/6 and, in combination with letrozole (Phase II; PALOMA-1) or with fulvestrant (Phase III; PALOMA-3), it has significantly prolonged progression-free survival, in patients with metastatic estrogen receptor-positive, HER2-negative breast cancer, in comparison with that observed in patients using letrozole, or fulvestrant alone, respectively. In this review, we provide an overview of the current compounds available for cell-cycle checkpoint protein-directed therapy for solid tumors. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells.

PubMed

Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

2015-04-10

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

PubMed

Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

2016-10-01

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.
Aqueous extract of Tribulus terrestris Linn induces cell growth arrest and apoptosis by down-regulating NF-κB signaling in liver cancer cells.

PubMed

Kim, Hye Jin; Kim, Jin Chul; Min, Jung Sun; Kim, Mi-Jee; Kim, Ji Ae; Kor, Myung Ho; Yoo, Hwa Seung; Ahn, Jeong Keun

2011-06-14

A medicinal herb Tribulus terrestris Linn has been used to treat various diseases including hepatocellular carcinoma. The aim of the present study was to investigate the anticancer activity of Tribulus terrestris Linn (TT) in liver cancer cells. The antitumor activity of aqueous TT extract was analyzed by testing the cytotoxicity and the effect on clonogenecity in HepG2 cells. Apoptosis and cell cycle arrest induced by TT were dissected by flow cytometry and its inhibitory effect on NF-κB activity was determined by analyzing the expression levels of NF-κB/IκB subunit proteins. The suppression of NF-κB-regulated gene expression by TT was assessed by RT-PCR. TT extract repressed clonogenecity and proliferation, induced apoptosis, and enhanced accumulation in the G0/G1 phase of liver cancer cells. It also turned out that TT extract inhibited NF-κB-dependent reporter gene expression and NF-κB subunit p50 expression, while it enhanced the cellular level of IκBα by inhibiting the phosphorylation and degradation of IκBα. In addition, IKK activity was inhibited in a dose-dependent manner. Furthermore, TT extract suppressed the transcription of genes associated with cell cycle regulation, anti-apoptosis, and invasion. These data showed that TT extract blocks proliferation and induces apoptosis in human liver cancer cells through the inhibition of NF-κB signaling. Aqueous TT extract can be used as an anticancer drug for hepatocellular carcinoma patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Metabolic effect of TAp63α: enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense

PubMed Central

D'Alessandro, Angelo; Amelio, Ivano; Berkers, Celia R.; Antonov, Alexey; Vousden, Karen H.; Melino, Gerry; Zolla, Lello

2014-01-01

TAp63α is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation. In order to assess whether TAp63α plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in which the expression of TAp63α was dependent on doxycycline supplementation to the medium. Metabolomics labeling experiments were performed by incubating the cells in 13C-glucose or 13C15N-glutamine-labeled culture media, as to monitor metabolic fluxes upon induced expression of TAp63α. Induced expression of TAp63α resulted in cell cycle arrest at the G1 phase. From a metabolic standpoint, expression of Tap63α promoted glycolysis and the pentose phosphate pathway, which was uncoupled from nucleotide biosynthesis, albeit prevented oxidative stress in the form of oxidized glutathione. Double 13C-glucose and 13C15N-glutamine metabolic labeling confirmed that induced expression of TAp63α corresponded to a decreased flux of pyruvate to the Krebs cycle and decreased utilization of glutamine for catabolic purposes in the TCA cycle. Results were not conclusive in relation to anabolic utilization of labeled glutamine, since it is unclear to what extent the observed minor TAp63α-dependent increases of glutamine-derived labeling in palmitate could be tied to increased rates of reductive carboxylation and de novo synthesis of fatty acids. Finally, bioinformatics elaborations highlighted a link between patient survival rates and the co-expression of p63 and rate limiting enzymes of the pentose phosphate pathway, G6PD and PGD. PMID:25229745
In vitro and in vivo anti-tumour activities of echinoside A and ds-echinoside A from Pearsonothuria graeffei.

PubMed

Zhao, Qin; Xue, Yong; Wang, Jing-feng; Li, Hui; Long, Teng-teng; Li, Zhaojie; Wang, Yu-ming; Dong, Ping; Xue, Chang-hu

2012-03-15

Echinoside A (EA) and ds-echinoside A (DSEA) are triterpene glycosides isolated from the sea cucumber Pearsonothuria graeffei. DSEA, the desulfurisation product of EA, has the following structure: β-D-xylopyranosyl-holost-8(9),11(12)-diene-3β,17α-diol. In the present study, we examined the anti-tumour activities-in particular, the structure-activity relationships-of EA and DSEA in vitro and in vivo. Both EA and DSEA exhibited an inhibitory effect on cell proliferation, along with apoptosis-inducing activity, in HepG2 cells. Moreover, they significantly arrested the cell cycle in the G₀/G₁ phase. A reverse transcriptase-polymerase chain reaction assay revealed that EA and DSEA significantly increased the expression of the cell-cycle-related genes, namely, p16, p21 and c-myc, and decreased that of cyclin D₁. Western blotting analysis demonstrated that they down-regulated the expression of Bcl-2, and enhanced mitochondria cytochrome c release, caspase-3 activation, and poly(adenosine diphosphate ribose) polymerase, cleavage. Nuclear factor kappa B (NF-κB) expression was significantly decreased by DSEA, but was unaffected by EA. EA and DSEA (2.5 mg kg⁻¹) treatment of mice bearing H22 hepatocarcinoma tumours reduced the tumour weight by 49.8% and 55.0%, respectively. EA and DSEA exhibit marked anti-cancer activity in HepG2 cells, by blocking cell-cycle progression and inducing apoptosis through the mitochondrial pathway. DSEA-induced apoptosis was more potent than EA-induced apoptosis. Furthermore, the two triterpene glycosides derived from P. graeffei may induce apoptosis of HepG2 cells in an NF-κB-dependent or NF-κB-independent manner, depending on their structure. Copyright © 2011 Society of Chemical Industry.
The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth.

PubMed

Palazzo, E; Kellett, M; Cataisson, C; Gormley, A; Bible, P W; Pietroni, V; Radoja, N; Hwang, J; Blumenberg, M; Yuspa, S H; Morasso, M I

2016-06-16

Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinguished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis.
The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth

PubMed Central

Palazzo, Elisabetta; Kellett, Meghan; Cataisson, Christophe; Gormley, Anna; Bible, Paul W.; Pietroni, Valentina; Radoja, Nadezda; Hwang, Joonsung; Blumenberg, Miroslav; Yuspa, Stuart H.; Morasso, Maria

2015-01-01

Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinquished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis. PMID:26522723
Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

PubMed Central

Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

2013-01-01

Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816
DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinotti, Simona; Ranzato, Elia, E-mail: ranzato@unipmn.it; Parodi, Monica

Malignant mesothelioma (MMe) is a poor-prognosis tumor in need of innovative therapies. In a previous in vivo study, we showed synergistic anti-MMe properties of the ascorbate/epigallocatechin-3-gallate/gemcitabine combination. We have now focused on the mechanism of action, showing the induction of apoptosis and cell cycle arrest through measurements of caspase 3, intracellular Ca{sup 2+}, annexin V, and DNA content. StellArray™ PCR technology and Western immunoblotting revealed DAPK2-dependent apoptosis, upregulation of cell cycle promoters, downregulation of cell cycle checkpoints and repression of NFκB expression. The complex of data indicates that the mixture is synergistic in inducing cell cycle deregulation and non-inflammatory apoptosis,more » suggesting its possible use in MMe treatment. - Highlights: • Ascorbate/epigallocathechin-gallate/gemcitabine has been tested on mesothelioma cells • A synergistic mechanism has been shown for cell cycle arrest and apoptosis • PCR-array analysis has revealed the de-regulation of apoptosis and cell cycle genes • Maximum upregulation has been found for the Death-Associated Protein Kinase-2 gene • Data suggest that the mixture could be used as a clinical treatment.« less
Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

NASA Astrophysics Data System (ADS)

Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p53R2 gene expression modulations shows a response lasting up to 24 hours after irradiation.
Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

PubMed

Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

2004-06-18

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.
Inhibition of Midkine Suppresses Prostate Cancer CD133+ Stem Cell Growth and Migration.

PubMed

Erdogan, Suat; Doganlar, Zeynep B; Doganlar, Oguzhan; Turkekul, Kader; Serttas, Riza

2017-09-01

Midkine (MDK) is a tumor-promoting factor that is often overexpressed in various human carcinomas, and the role of MDK has not yet been fully investigated in prostate cancer stem cells. Prostate cancer CD133 + stem cells (PCSCs) were isolated from human castration-resistant PC3 cells. PCSCs were treated with different concentrations of MDK inhibitor, iMDK, for 24-72 hours. The IC 50 values were determined by the MTT test. Endogenous MDK messenger RNA expression was knocked down by small interfering RNA. Quantitative reverse transcription polymerase chain reaction, Western blot analyses and image-based cytometry were used to investigate apoptosis and cell cycle progression as well as their underlying molecular mechanisms. Cell migration was evaluated by the wound healing test. iMDK caused dose- and time-dependent inhibition of PCSC survival. Similar growth inhibition was also obtained by small interfering RNA-mediated knockdown of endogenous MDK expression. iMDK was shown to preferentially induce cell cycle arrest at the S and G2/M phases. Suppressed PCSC growth was also accompanied by increases in p53 and the cell cycle inhibitor p21 genes. Combinatorial treatment of iMDK with docetaxel significantly inhibited cell proliferation versus either of the agents used alone. Inhibition of MDK expression strongly suppressed the migration of PCSCs compared to untreated and docetaxel-treated cells. iMDK and the knockdown of MDK decreased p-Akt and significantly upregulated the expression of PI3K/phosphatase/tensin homolog. Our data indicate that MDK plays a crucial role in controlling PCSC proliferation and migration. Therefore, suppression of endogenous expression of MDK would, in combination with traditional chemotherapy drugs, be a potential treatment for PCSCs. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.
α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells.

PubMed

Lin, Jen-Jyh; Yu, Chien-Chih; Lu, Kung-Wen; Chang, Shu-Jen; Yu, Fu-Shun; Liao, Ching-Lung; Lin, Jaung-Geng; Chung, Jing-Gung

2014-08-01

α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Global gene expression analysis combined with a genomics approach for the identification of signal transduction networks involved in postnatal mouse myocardial proliferation and development.

PubMed

Wang, Ruoxin; Su, Chao; Wang, Xinting; Fu, Qiang; Gao, Xingjie; Zhang, Chunyan; Yang, Jie; Yang, Xi; Wei, Minxin

2018-01-01

Mammalian cardiomyocytes may permanently lose their ability to proliferate after birth. Therefore, studying the proliferation and growth arrest of cardiomyocytes during the postnatal period may enhance the current understanding regarding this molecular mechanism. The present study identified the differentially expressed genes in hearts obtained from 24 h‑old mice, which contain proliferative cardiomyocytes; 7‑day‑old mice, in which the cardiomyocytes are undergoing a proliferative burst; and 10‑week‑old mice, which contain growth‑arrested cardiomyocytes, using global gene expression analysis. Furthermore, myocardial proliferation and growth arrest were analyzed from numerous perspectives, including Gene Ontology annotation, cluster analysis, pathway enrichment and network construction. The results of a Gene Ontology analysis indicated that, with increasing age, enriched gene function was not only associated with cell cycle, cell division and mitosis, but was also associated with metabolic processes and protein synthesis. In the pathway analysis, 'cell cycle', proliferation pathways, such as the 'PI3K‑AKT signaling pathway', and 'metabolic pathways' were well represented. Notably, the cluster analysis revealed that bone morphogenetic protein (BMP)1, BMP10, cyclin E2, E2F transcription factor 1 and insulin like growth factor 1 exhibited increased expression in hearts obtained from 7‑day‑old mice. In addition, the signal transduction pathway associated with the cell cycle was identified. The present study primarily focused on genes with altered expression, including downregulated anaphase promoting complex subunit 1, cell division cycle (CDC20), cyclin dependent kinase 1, MYC proto-oncogene, bHLH transcription factor and CDC25C, and upregulated growth arrest and DNA damage inducible α in 10-week group, which may serve important roles in postnatal myocardial cell cycle arrest. In conclusion, these data may provide important information regarding myocardial proliferation and development.
Sulforaphane inhibits TGF-β-induced epithelial-mesenchymal transition of hepatocellular carcinoma cells via the reactive oxygen species-dependent pathway.

PubMed

Wu, Jinsheng; Han, Jingli; Hou, Benxin; Deng, Chengwei; Wu, Huanliang; Shen, Liangfang

2016-05-01

Sulforaphane is recognized as a safe antitumor agent derived from various cruciferous vegetables, including broccoli. It has been demonstrated that sulforaphase is a potent antitumor agent in diverse cancers. However, its effect on hepatocellular carcinoma remains largely unknown. Here, we show that sulforaphane inhibits TGF-β-induced epithelial-mesenchymal transition of hepatocellular carcinoma cell via the reactive oxygen species-dependent pathway. We found sulforaphane inhibited hepatocellular carcinoma cell proliferation in a dose- and time-dependent manner. Sulforaphane induced G0/G1 phase cell cycle arrest and promoted cell apoptosis. A set of experiments showed that sulforaphase inhibited hepatocellular carcinoma cell migration and invasion, inhibited the formation of fibroblast like mesenchymal cells and the expression of Vimentin, but increased the expression of E-cadherin, suggesting sulforaphane suppresses epithelial-mesenchymal transition (EMT) process. Cotreatment with N-acetyl-L-cysteine inhibited sulforaphane-inhibited invasion and upregulation of E-cadherin and almost completely abolished the sulforaphane-induced expression of Vimentin. The effect of sulforaphane on the growth of hepatocellular carcinoma cells was confirmed by a xenograft tumor growth model. All our finding indicated that sulforaphane is a promising and safe strategy for treating hepatocellular carcinoma.
Piperlongumine decreases cell proliferation and the expression of cell cycle-associated proteins by inhibiting Akt pathway in human lung cancer cells.

PubMed

Seok, Jin Sil; Jeong, Chang Hee; Petriello, Michael C; Seo, Han Geuk; Yoo, Hyunjin; Hong, Kwonho; Han, Sung Gu

2018-01-01

Piperlongumine (PL) is an alkaloid of a pepper plant found in Southeast Asia. PL is known to induce selective toxicity towards a variety of cancer cell types. To explore the possible anti-lung cancer effects of PL, A549 cells were treated with PL (0-40 μM) for 24 h. Alterations in the expression of cell cycle-associated proteins (cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb)) and intracellular signaling molecules (extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, p38 and nuclear factor-κB (NF-κB)) were examined in cells following treatment of PL using Western blot analysis. Results showed that proliferation of cells were significantly decreased by PL in a dose-dependent manner. Flow cytometry results demonstrated increased number of cells in G1 phase in PL (40 μM)-treated group. Reactive oxygen species was significantly increased in cells treated with PL at 20-40 μM. The expression of cyclin D1, CDK4, CDK6 and p-Rb were markedly decreased in cells treated with PL at 40 μM. Treatment of cells with PL suppressed phosphorylation of Akt but increased ERK1/2 phosphorylation. Treatment of PL significantly decreased nuclear translocation of NF-κB p65 in cells. These results suggest that PL possesses antiproliferative properties in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.
Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

PubMed Central

1996-01-01

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331
Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

PubMed Central

Deniaud, Emmanuelle; Baguet, Joël; Chalard, Roxane; Blanquier, Bariza; Brinza, Lilia; Meunier, Julien; Michallet, Marie-Cécile; Laugraud, Aurélie; Ah-Soon, Claudette; Wierinckx, Anne; Castellazzi, Marc; Lachuer, Joël; Gautier, Christian

2009-01-01

Background The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. Methodology and Principal Findings We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. Conclusion This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms. PMID:19753117
Discovery of time-delayed gene regulatory networks based on temporal gene expression profiling

PubMed Central

Li, Xia; Rao, Shaoqi; Jiang, Wei; Li, Chuanxing; Xiao, Yun; Guo, Zheng; Zhang, Qingpu; Wang, Lihong; Du, Lei; Li, Jing; Li, Li; Zhang, Tianwen; Wang, Qing K

2006-01-01

Background It is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks. Results In particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings. Conclusion We established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to the development, aging and progressive pathogenesis of a complex disease where potential dependences between different experiment units might occurs. PMID:16420705
The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525
The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

PubMed

Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.
Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

PubMed

Han, Guocan; Zhou, Qiang

2016-12-01

Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P < 0.05). The apoptotic HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P < 0.05). Meanwhile, loss of △Ψm, increase in reactive oxygen species and O 2 ·- , and the decrease in catalase activity and glutathione (GSH) level were found after DMF treatment (P < 0.05). All these changes were significantly attenuated and even completely disappeared by adding NAC (P < 0.05). In conclusion, the cytotoxicity of DMF on cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.
AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.
Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

PubMed Central

Patsoukis, Nikolaos; Brown, Julia; Petkova, Victoria; Liu, Fang; Li, Lequn; Boussiotis, Vassiliki A.

2017-01-01

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle. PMID:22740686
Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kito, Hiroaki; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto; Yamamura, Hisao

2015-04-10

Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cyclemore » progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.« less
Pause, play, repeat

PubMed Central

Sansó, Miriam; Fisher, Robert P

2013-01-01

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3′ end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the “beginning” and “end” of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3′ end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research. PMID:23756342
Circadian clock regulation of the cell cycle in the zebrafish intestine.

PubMed

Peyric, Elodie; Moore, Helen A; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.
Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

PubMed Central

Peyric, Elodie; Moore, Helen A.; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally. PMID:24013905
Cyclin-dependent kinase inhibitor Cdkn2c regulates B cell homeostasis and function in the NZM2410-derived murine lupus susceptibility locus Sle2c1

PubMed Central

Xu, Zhiwei; Potula, Hari-Hara SK; Vallurupalli, Anusha; Perry, Daniel; Baker, Henry; Croker, Byron P.; Dozmorov, Igor; Morel, Laurence

2013-01-01

Sle2c1 is an NZM2410 and NZB-derived lupus susceptibility locus that induces an expansion of the B1a cell compartment. B1a cells have a repertoire enriched for autoreactivity, and an expansion of this B cell subset occurs in several mouse models of lupus. A combination of genetic mapping and candidate gene analysis presents Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18INK4c (p18), as the top candidate gene for inducing the Slec2c1 associated expansion of B1a cells. A novel SNP in the NZB allele of the Cdkn2c promoter is associated with a significantly reduced Cdkn2c expression in the splenic B cells and Pc B1a cells from Sle2c1-carrying mice, which leads to a defective G1 cell cycle arrest in splenic B cells and increased proliferation of Pc B1a cells. As cell cycle is differentially regulated in B1a and B2 cells, these results suggest that Cdkn2c plays a critical role in B1a cell self-renewal, and that its impaired expression leads to an accumulation of these cells with high autoreactive potential. PMID:21543644
Sarcosine Up-Regulates Expression of Genes Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer

PubMed Central

Heger, Zbynek; Merlos Rodrigo, Miguel Angel; Michalek, Petr; Polanska, Hana; Masarik, Michal; Vit, Vitezslav; Plevova, Mariana; Pacik, Dalibor; Eckschlager, Tomas; Stiborova, Marie

2016-01-01

The effects of sarcosine on the processes driving prostate cancer (PCa) development remain still unclear. Herein, we show that a supplementation of metastatic PCa cells (androgen independent PC-3 and androgen dependent LNCaP) with sarcosine stimulates cells proliferation in vitro. Similar stimulatory effects were observed also in PCa murine xenografts, in which sarcosine treatment induced a tumor growth and significantly reduced weight of treated mice (p < 0.05). Determination of sarcosine metabolism-related amino acids and enzymes within tumor mass revealed significantly increased glycine, serine and sarcosine concentrations after treatment accompanied with the increased amount of sarcosine dehydrogenase. In both tumor types, dimethylglycine and glycine-N-methyltransferase were affected slightly, only. To identify the effects of sarcosine treatment on the expression of genes involved in any aspect of cancer development, we further investigated expression profiles of excised tumors using cDNA electrochemical microarray followed by validation using the semi-quantitative PCR. We found 25 differentially expressed genes in PC-3, 32 in LNCaP tumors and 18 overlapping genes. Bioinformatical processing revealed strong sarcosine-related induction of genes involved particularly in a cell cycle progression. Our exploratory study demonstrates that sarcosine stimulates PCa metastatic cells irrespectively of androgen dependence. Overall, the obtained data provides valuable information towards understanding the role of sarcosine in PCa progression and adds another piece of puzzle into a picture of sarcosine oncometabolic potential. PMID:27824899
Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

PubMed

Fernández, Ignacio; Vijayakumar, Parameswaran; Marques, Carlos; Cancela, M Leonor; Gavaia, Paulo J; Laizé, Vincent

2015-06-01

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.
Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology.

PubMed

Dutto, Ilaria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A; Prosperi, Ennio

2015-02-01

The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.
Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation

PubMed Central

Kim, Ji Hyun; Ki, Soo Mi; Joung, Je-Gun; Scott, Eric; Heynen-Genel, Susanne; Aza-Blanc, Pedro; Kwon, Chang Hyuk; Kim, Joon; Gleeson, Joseph G.; Lee, Ji Eun

2016-01-01

Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as AURKA and PLK1, and 2) the ubiquitin-proteasome systems (UPS)-involved hits are necessary for proteolysis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum withdrawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry. PMID:27033521
Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways.

PubMed

Chen, Yi-Jin; Wang, Wen-Hung; Wu, Wan-Yu; Hsu, Chia-Chi; Wei, Ling-Rung; Wang, Sheng-Fan; Hsu, Ya-Wen; Liaw, Chih-Chuang; Tsai, Wan-Chi

2017-01-01

Pancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer. Human pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 in vivo was evaluated by utilizing BxPC-3 xenograft mouse model. AR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth in vivo. AR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors in vivo. We conclude that by virtue of its multiple mechanisms of action, AR-42 possesses a considerable potential as an antitumor agent in pancreatic cancer.
Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229
Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.
The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

PubMed Central

Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

2015-01-01

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues. PMID:26067441
Molecular Genetic Traits Influencing Maize Endosperm Development and Value: Closeout Report for DOE Grant DE-FG02-96ER20242

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brian A. Larkins

2012-09-12

Development of the endosperm in cereal grasses entails different phases characterized by cell division, endoreduplication, accumulation of storage metabolites and cell death, which need to be carried out in an orderly fashion. While correct regulation of the cell cycle plays an essential role in endosperm development, the key regulatory factors and how the cell cycle interfaces with other pathways in this developmental context are largely unknown. We investigated the cyclin-dependent kinase (CDK)-retinoblastoma pathway and how it controls the cell cycle and coordinates it with other processes during maize endosperm development. Retinoblastoma-related (RBR) proteins may be inactivated through CDK-mediated phosphorylation, butmore » the identity of the responsible kinase in maize is unknown. We have previously shown that down-regulation of CDKA;1 severely inhibits the endoreduplication cell cycle and suggested that CDK may be an up-stream regulator of the retinoblastoma pathway. We discovered two types of maize RBR genes, RBR1 and RBR3, which differ in terms of structure, regulation and function. Phylogenetic analyses indicate that these genes may be distinctive features of the Poaceae. We found that RBR3 plays a positive rather than a negative role in DNA replication, cell transformation, and the expression of the minichromosome maintenance (MCM)2-7 family of DNA replication factors. These features are a paradigm shift in RBR gene function and appear to be unique within the RBR gene family. They suggest the existence in maize and related cereal crops of specific RBR/E2F-dependent pathways impinging on the cell cycle and development. RBR1 was down-regulated in transgenic endosperm using RNAi approaches. This resulted in the de-repression of a number of down-stream E2F targets, including RBR3, the MCM2-7 gene family, DNA methyltransferase (MET)1, CDKB;1, and the recently identified RBR4 gene. It also increased endosperm ploidy levels, stimulated the production of a larger number of cells, reduced the average cell size, and promoted programmed cell death. To test whether CDKA;1 inhibits RBR1 (through phosphorylation) in the pathway that leads to DNA synthesis and endoreduplication, the two CDKA;1 and RBR1 down-regulated mutants were crossed and their progeny analyzed. Our results indicate that CDKA;1 controls endoreduplication through an RBR1-dependent pathway. However, the ability of RBR1 to repress gene expression programs is independent from CDKA1, suggesting the presence of two differently regulated RBR1 activities in developing endosperm. One type of RBR1 activity controls E2F-dependent gene expression and is largely independent from CDKA;1, while another suppresses endoreduplication and can be inhibited by CDKA;1. In addition, RBR1 is part of a regulatory feedback loop that impinges on CDK activity. Together, these results indicate that the CDKA;1-RBR1 pathway integrates and controls different processes associated with endosperm development. Genome-wide analyses of the transcriptome, metabolome, and epigenetic mechanisms to understand how the cell cycle is coordinated with other pathways at a systems biology level are currently underway.« less
A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.

PubMed

Schorpp, Kenji; Rothenaigner, Ina; Maier, Julia; Traenkle, Bjoern; Rothbauer, Ulrich; Hadian, Kamyar

2016-10-01

Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. © 2016 Society for Laboratory Automation and Screening.
Bax Translocation Mediated Mitochondrial Apoptosis and Caspase Dependent Photosensitizing Effect of Ficus religiosa on Cancer Cells

PubMed Central

Thankayyan R, Santhosh Kumar; Sithul, Hima; Sreeharshan, Sreeja

2012-01-01

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS. PMID:22792212

Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect.

PubMed

Reikvam, Håkon; Tamburini, Jerome; Skrede, Silje; Holdhus, Rita; Poulain, Laury; Ersvaer, Elisabeth; Hatfield, Kimberley J; Bruserud, Øystein

2014-01-01

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models. © 2013 John Wiley & Sons Ltd.
Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor

PubMed Central

Liu, Weijun; Xin, Hong; Eckert, David T.; Brown, Julie A.; Gnarra, James R.

2010-01-01

Inactivation of von Hippel-Lindau tumor suppressor protein (pVHL) is associated with von Hippel-Lindau disease, an inherited cancer syndrome, as well as the majority of patients with sporadic clear cell renal carcinoma (RCC). While the involvement of pVHL in oxygen sensing through targeting HIFα subunits to ubiquitin-dependent proteolysis has been well documented, less is known about pVHL regulation under both normoxic and hypoxic conditions. We found that pVHL levels decreased in hypoxia and that hypoxia-induced cell cycle arrest is associated with pVHL expression in RCC cells. pVHL levels fluctuate during the cell cycle, paralleling cyclin B1 levels, with decreased levels in mitosis and G1. pVHL contains consensus Destruction box sequences, and pVHL associates with Cdh1, an activator of the anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. We show that pVHL has a decreased half-life in G1, Cdh1 downregulation results in increased pVHL expression, while Cdh1 overexpression results in decreased pVHL expression. Taken together these results suggest that pVHL is a novel substrate of APC/CCdh1. Destruction box-independent pVHL degradation was also detected, indicating that other ubiquitin ligases are also activated for pVHL degradation. PMID:20802534
[1-9-NαC]-crourorb A1 isolated from Croton urucurana latex induces G2/M cell cycle arrest and apoptosis in human hepatocarcinoma cells.

PubMed

de Matos Cândido-Bacani, Priscila; Ezan, Frédéric; de Oliveira Figueiredo, Patrícia; Matos, Maria de Fátima Cepa; Rodrigues Garcez, Fernanda; Silva Garcez, Walmir; Baffet, Georges

2017-05-05

[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC 50 : 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality. Copyright © 2017 Elsevier B.V. All rights reserved.
RGC-32 is a novel regulator of the T-lymphocyte cell cycle.

PubMed

Tegla, Cosmin A; Cudrici, Cornelia D; Nguyen, Vinh; Danoff, Jacob; Kruszewski, Adam M; Boodhoo, Dallas; Mekala, Armugam P; Vlaicu, Sonia I; Chen, Ching; Rus, Violeta; Badea, Tudor C; Rus, Horea

2015-06-01

We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice developed normally and did not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells, we determined the proliferative rates of CD4(+) and CD8(+) T cells from the spleens of RGC-32(-/-) mice, as compared to wild-type (WT, RGC-32(+/+)) control mice. After stimulation with anti-CD3/anti-CD28, CD4(+) T cells from RGC-32(-/-) mice displayed a significant increase in [(3)H]-thymidine incorporation when compared to WT mice. In addition, both CD4(+) and CD8(+) T cells from RGC-32(-/-) mice displayed a significant increase in the proportion of proliferating Ki67(+) cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation induced by T-cell receptor/CD28 engagement. Furthermore, Akt and FOXO1 phosphorylation induced in stimulated CD4(+) T-cells from RGC-32(-/-) mice were significantly higher, indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. We also found that IL-2 mRNA and protein expression were significantly increased in RGC-32(-/-) CD4(+) T cells when compared to RGC-32(+/+) CD4(+) T cells. In addition, the effect of RGC-32 on the cell cycle and IL-2 expression was inhibited by pretreatment of the samples with LY294002, indicating a role for phosphatidylinositol 3-kinase (PI3K). Thus, RGC-32 is involved in controlling the cell cycle of T cells in vivo, and this effect is mediated by IL-2 in a PI3K-dependent fashion. Copyright © 2015 Elsevier Inc. All rights reserved.
Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival

PubMed Central

Thole, Theresa M; Lodrini, Marco; Fabian, Johannes; Wuenschel, Jasmin; Pfeil, Sebastian; Hielscher, Thomas; Kopp-Schneider, Annette; Heinicke, Ulrike; Fulda, Simone; Witt, Olaf; Eggert, Angelika; Fischer, Matthias; Deubzer, Hedwig E

2017-01-01

The number of long-term survivors of high-risk neuroblastoma remains discouraging, with 10-year survival as low as 20%, despite decades of considerable international efforts to improve outcome. Major obstacles remain and include managing resistance to induction therapy, which causes tumor progression and early death in high-risk patients, and managing chemotherapy-resistant relapses, which can occur years after the initial diagnosis. Identifying and validating novel therapeutic targets is essential to improve treatment. Delineating and deciphering specific functions of single histone deacetylases in neuroblastoma may support development of targeted acetylome-modifying therapeutics for patients with molecularly defined high-risk neuroblastoma profiles. We show here that HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs. Genes necessary for mitotic cell cycle progression and cell division were most prominently enriched in at least two of three time points in whole-genome expression data combined from two cell systems, and all nine genes in these functional categories were strongly repressed, including CENPA, KIF14, KIF23 and RACGAP1. Enforced expression of one selected candidate, RACGAP1, partially rescued the induction of apoptosis caused by HDAC11 depletion. High-level expression of all nine genes in primary neuroblastomas significantly correlated with unfavorable overall and event-free survival in patients, suggesting a role in mediating the more aggressive biological and clinical phenotype of these tumors. Our study identified a group of cell cycle-promoting genes regulated by HDAC11, being both predictors of unfavorable patient outcome and essential for tumor cell viability. The data indicate a significant role of HDAC11 for mitotic cell cycle progression and survival of MYCN-amplified neuroblastoma cells, and suggests that HDAC11 could be a valuable drug target. PMID:28252645
Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

PubMed

Ezhilarasan, Devaraj; Evraerts, Jonathan; Sid, Brice; Calderon, Pedro Buc; Karthikeyan, Sivanesan; Sokal, Etienne; Najimi, Mustapha

2017-02-01

Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 μmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.
Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells

PubMed Central

Hong, Heeok; An, Jeong Cheol; de La Cruz, Joseph F.; Hwang, Seong-Gu

2017-01-01

A number of diverse studies have reported the anticancer properties of Cnidium officinale Makino (CO). However, the apoptotic effect of this traditional medicinal herb in human hepatocellular carcinoma cells (HepG2) remains to be elucidated. Therefore, the present study investigated the ability of CO to reduce cell viability through apoptotic pathways. Cell viability was determined using the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay. CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cell cycle was monitored using fluorescence-activated cell sorting analysis with propidium iodide staining. Furthermore, the present study explored whether various signaling molecules associated with HepG2 cell death were affected by CO treatment, including caspase-3, B-cell lymphoma 2 (Bcl-2), tumor protein p53 (p53), cyclin-dependent kinase 4 (CDK4) and cyclin D. The expression levels of these genes were examined by reverse-transcription polymerase chain reaction and western blotting. The expression levels of caspase-3 and p53 were upregulated with CO extract treatment, whereas those of Bcl-2, CDK4 and cyclin D were significantly downregulated. Cleaved caspase-3 expression was upregulated following treatment with CO extract in a dose-dependent manner. Collectively, the data suggest that CO extract has the potential to induce apoptosis of HepG2 cells and may act by suppressing the cell cycle, which leads to caspase-3 cleavage and p53 signaling. PMID:28966688
ATM Expression Predicts Veliparib and Irinotecan Sensitivity in Gastric Cancer by Mediating P53-Independent Regulation of Cell Cycle and Apoptosis.

PubMed

Subhash, Vinod Vijay; Tan, Shi Hui; Yeo, Mei Shi; Yan, Fui Leng; Peethala, Praveen C; Liem, Natalia; Krishnan, Vaidehi; Yong, Wei Peng

2016-12-01

Identification of synthetically lethal cellular targets and synergistic drug combinations is important in cancer chemotherapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signaling and cell-cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its implications as a predictive biomarker for cancer chemotherapy remain unexplored. The present study evaluated ATM-induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, veliparib (ABT-888) and irinotecan (CPT-11), respectively. ATM expression was detected in a panel of gastric cell lines, and the IC 50 against each inhibitors was determined. The combinatorial effect of ABT-888 and CPT-11 in gastric cancer cells was also determined both in vitro and in vivo ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy; however, they showed a higher therapeutic effect with ABT-888 and CPT-11 combinatorial therapy. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell-cycle progression and apoptosis in a P53-independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cells to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM-dependent regulation of cellular processes. Mol Cancer Ther; 15(12); 3087-96. ©2016 AACR. ©2016 American Association for Cancer Research.
Research Resource: Global Identification of Estrogen Receptor β Target Genes in Triple Negative Breast Cancer Cells

PubMed Central

Shanle, Erin K.; Zhao, Zibo; Hawse, John; Wisinski, Kari; Keles, Sunduz; Yuan, Ming

2013-01-01

Breast cancers that are negative for estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 are known as triple-negative breast cancers (TNBC). TNBCs are associated with an overall poor prognosis because they lack expression of therapeutic targets like ERα and are biologically more aggressive. A second estrogen receptor, ERβ, has been found to be expressed in 50% to 90% of ERα-negative breast cancers, and ERβ expression in TNBCs has been shown to correlate with improved disease-free survival and good prognosis. To elucidate the role of ERβ in regulating gene expression and cell proliferation in TNBC cells, the TNBC cell line MDA-MB-468 was engineered with inducible expression of full-length ERβ. In culture, ERβ expression inhibited cell growth by inducing a G1 cell cycle arrest, which was further enhanced by 17β-estradiol treatment. In xenografts, ERβ expression also inhibited tumor formation and growth, and 17β-estradiol treatment resulted in rapid tumor regression. Furthermore, genomic RNA sequencing identified both ligand-dependent and -independent ERβ target genes, some of which were also regulated by ERβ in other TNBC cell lines and correlated with ERβ expression in a cohort of TNBCs from the Cancer Genome Atlas Network. ERβ target genes were enriched in genes that regulate cell death and survival, cell movement, cell development, and growth and proliferation, as well as genes involved in the Wnt/β-catenin and the G1/S cell cycle phase checkpoint pathways. In addition to confirming the anti-proliferative effects of ERβ in TNBC cells, these data provide a comprehensive resource of ERβ target genes and suggest that ERβ may be targeted with ligands that can stimulate its growth inhibitory effects. PMID:23979844
Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis

PubMed Central

Kim, Hyoji; Choi, Hoyun

2015-01-01

ABSTRACT Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. IMPORTANCE EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis. PMID:26581978
DOE Office of Scientific and Technical Information (OSTI.GOV)

Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferationmore » and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080 cells growth.« less
Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

PubMed

Liu, Jin-Yun; Liu, Zhong; Wang, Dong-Mei; Li, Man-Mei; Wang, Shao-Xiang; Wang, Rui; Chen, Jian-Ping; Wang, Yi-Fei; Yang, De-Po

2011-04-25

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523
Indigo naturalis and its component tryptanthrin exert anti-angiogenic effect by arresting cell cycle and inhibiting Akt and FAK signaling in human vascular endothelial cells.

PubMed

Chang, Hsin-Ning; Huang, Sheng-Teng; Yeh, Yuan-Chieh; Wang, Hsin-Shih; Wang, Tzu-Hao; Wu, Yi-Hong; Pang, Jong-Hwei S

2015-11-04

Indigo naturalis has been used to treat inflammatory diseases and dermatosis, including psoriasis, since thousands of years in China. It has been proven effective in our previous clinical studies on treating psoriasis, but the active component and the mechanism of how indigo naturalis working still needs to be clarified. Since the dysregulated angiogenesis is known to play an important role in the pathogenesis of psoriasis, the anti-angiogenic effect of indigo naturalis and tryptanthrin, a pure component of indigo naturalis, was investigated. The in vivo angiogenesis was studied by chick chorioallantoic membrane assay. The in vitro studies were performed using human vascular endothelial cells. Cell viability was determined by MTT assay. Cell cycle distribution was revealed by flow cytometry. The cellular messenger (m)RNA or protein expression level was analyzed by real-time RT-PCR or Western blot, respectively. Transwell filter migration assay and matrix gel-induced tube formation method were applied to examine the angiogenic potential. Indigo naturalis significantly inhibited the in vivo vascular endothelial growth factor (VEGF)-induced angiogenesis, as well as tryptanthrin. In vitro studies confirmed that indigo naturalis and tryptanthrin reduced the number of viable vascular endothelial cells. Tryptanthrin resulted in a cell cycle arrest and dose-dependently decreased the expressions of cyclin A, cyclin B, cyclin dependent kinase(CDK) 1 and 2, but not cyclin D and cyclin E, at both the mRNA and protein levels. The migration and tube formation of vascular endothelial cells were significantly inhibited by tryptanthrin in a dose-dependent manner. Result also showed that tryptanthrin could reduce the phosphorylated levels of both protein kinase B (PKB or Akt) and focal adhesion kinase (FAK). All together, these results demonstrated the anti-angiogenic effect of tryptanthrin, the acting component of indigo naturalis and revealed the underlying mechanism by inhibiting the cell cycle progression, cell migration and tube formation, likely mediated through blocking the Akt and FAK pathways. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

PubMed Central

Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

2015-01-01

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways. PMID:25632222
Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation

PubMed Central

Ankers, John M; Awais, Raheela; Jones, Nicholas A; Boyd, James; Ryan, Sheila; Adamson, Antony D; Harper, Claire V; Bridge, Lloyd; Spiller, David G; Jackson, Dean A; Paszek, Pawel; Sée, Violaine; White, Michael RH

2016-01-01

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.10473.001 PMID:27185527
Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

PubMed

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.
FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways.

PubMed

Kelleher, Fergal C; O'Sullivan, Hazel

2016-07-05

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin - cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome.FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of β-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy.
Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

PubMed

Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

2004-09-03

Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.
Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

PubMed

Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells.

PubMed

Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

2011-10-01

Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G(1) to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.
Cell cycle pathway dysregulation in human keratinocytes during chronic exposure to low arsenite.

PubMed

Al-Eryani, Laila; Waigel, Sabine; Jala, Venkatakrishna; Jenkins, Samantha F; States, J Christopher

2017-09-15

Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point. HaCaT cells were exposed to 0 or 100nM NaAsO 2 for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry. 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells. Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes. Copyright © 2017 Elsevier Inc. All rights reserved.
Abnormal Activation of RhoA/ROCK-I Signaling in Junctional Zone Smooth Muscle Cells of Patients With Adenomyosis.

PubMed

Wang, S; Duan, H; Zhang, Y; Sun, F Q

2016-03-01

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. The RhoA/Rho-kinase (ROCK) signaling pathway is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. Here we examined the potential role of this pathway in junctional zone (JZ) contraction in women with and without ADS. We demonstrated that in the normal JZ, RhoA and ROCK-I messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. Expression of RhoA and ROCK-I in the JZ from women with ADS was significantly higher than in the control women and showed no significant differences across the menstrual cycle. Treatment of JZ smooth muscle cells (JZSMCs) with estrogen at 0, 1, 10, or 100 nmol/L for 24 hours resulted in increased expression of RhoA, ROCK-I, and myosin light-chain (MLC) phosphorylation (p-MLC) in a dose-dependent manner. In parallel to its effects on p-MLC, estrogen-mediated, dose-dependent contraction responses in JZSMCs. Estrogen-mediated contraction in the ADS group was significantly higher than in the controls and also showed no significant differences across the menstrual cycle. These effects were suppressed in the presence of ICI 182780 or Y27632, supporting an estrogen receptor-dependent and RhoA activation-dependent mechanism. Our results indicate that the level of RhoA and ROCK-I increases in patients with ADS and the cyclic change is lost. Estrogen may affect uterine JZ contraction of ADS by enhancing RhoA/ ROCK-I signaling. © The Author(s) 2015.
Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

PubMed Central

Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

2011-01-01

The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652
Coordination of the cell cycle is an important determinant of the syndrome of acute renal failure.

PubMed

Megyesi, Judit; Andrade, Lucia; Vieira, Jose M; Safirstein, Robert L; Price, Peter M

2002-10-01

Recovery from injury is usually accompanied by cell replication, in which new cells replace those irreparably damaged. After acute renal failure, normally quiescent kidney cells enter the cell cycle, which in tubule segments is accompanied by the induction of cell cycle inhibitors. We found that after acute renal failure induced by either cisplatin injection or renal ischemia, induction of the p21 cyclin-dependent kinase (cdk) inhibitor is protective. Mice lacking this gene developed more widespread kidney cell death, more severe renal failure, and had reduced survival, compared with mice with a functional p21 gene. Here, we show induction of 14-3-3sigma, a regulator of G(2)-to-M transition, after acute renal failure. Our findings, using both in vivo and in vitro models of acute renal failure, show that this protein likely helps to coordinate cell cycle activity to maximize recovery of renal epithelial cells from injury and reduce the extent of the injury itself. Because in terminally differentiated cells, these proteins are highly expressed only after injury, we propose that cell cycle coordination by induction of these proteins could be a general model of tissue recovery from stress and injury.
Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

PubMed

Bristow, Sara L; Leman, Adam R; Simmons Kovacs, Laura A; Deckard, Anastasia; Harer, John; Haase, Steven B

2014-09-05

The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.
Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

PubMed Central

2011-01-01

Background Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD. Results Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition. Conclusions The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis. PMID:22112898
Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367
Expression of Genes Associated with DNA Damage Sensing in Human Fibroblasts Exposed to Low-dose-rate Gamma Rays

NASA Technical Reports Server (NTRS)

Zhang, Ye; Mehta, Satish; Hammond, Diane; Pierson, Duane; Jeevarajan, Antony; Cucinotta, Francis; Rohde, Larry; Wu, Honglu

2007-01-01

Understanding of the molecular response to low-dose and low-dose-rate radiation exposure is essential for the extrapolation of high-dose radiation risks to those at dose levels relevant to space and other environmental concerns. Most of the reported studies of gene expressions induced by low-dose or low-dose-rate radiation were carried out on exponentially growing cells. In the present study, we analyzed expressions of 84 genes associated with DNA damage sensing using real time PCR in human fibroblasts in mostly G1 phase of the cell cycle. The cells were exposed continuously to gamma rays at a dose rate of 0.8 cGy/hr for 1, 2, 6 or 24 hrs at 37 C throughout the exposure. The total RNA was isolated immediately after the exposure was terminated. Of the 84 genes, only a few showed significant changes of the expression level. Some of the genes (e.g. DDit3 and BTG2) were found to be up or down regulated only after a short period of exposure, while other genes (e.g. PRKDC) displayed a highest expression level at the 24 hr time point. The expression profiles for the exposed cells which had a smaller portion of G1 cells indicated more cell cycle signaling and DNA repair genes either up or down regulated. Interestingly, the panel of genes changed from radiation exposure in G1 cells is different from the panel in cells having less G1 arrest cells. The gene expression profile of the cells responding to low-dose-radiation insult apparently depends on the cell growth stage. The response pathway in G1 cells may differ from that in exponentially growing cells.
The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells.

PubMed

Park, Eun Young; Lee, Kyung-Won; Lee, Heon-Woo; Cho, Young-Wuk; Baek, Nam-In; Chung, Hae-Gon; Jeong, Tae-Sook; Choi, Myung-Sook; Lee, Kyung-Tae

2008-06-01

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Peek, Gregory W.; Tollefsbol, Trygve O., E-mail: trygve@uab.edu; Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmitmore » signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression. - Highlights: • PX-866 and raloxifene affect the PI3K/Akt and TGF-β pathways. • PX-866 and raloxifene down-regulate genes up-regulated in cancer. • PX-866 and raloxifene decrease transcription of hTERT and Cyclin D1. • Pathological transcription signatures can identify new defense mechanisms.« less
Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.
Dynamics of gene expression with positive feedback to histone modifications at bivalent domains

NASA Astrophysics Data System (ADS)

Huang, Rongsheng; Lei, Jinzhi

2018-03-01

Experiments have shown that in embryonic stem cells, the promoters of many lineage-control genes contain “bivalent domains”, within which the nucleosomes possess both active (H3K4me3) and repressive (H3K27me3) marks. Such bivalent modifications play important roles in maintaining pluripotency in embryonic stem cells. Here, to investigate gene expression dynamics when there are regulations in bivalent histone modifications and random partition in cell divisions, we study how positive feedback to histone methylation/demethylation controls the transition dynamics of the histone modification patterns along with cell cycles. We constructed a computational model that includes dynamics of histone marks, three-stage chromatin state transitions, transcription and translation, feedbacks from protein product to enzymes to regulate the addition and removal of histone marks, and the inheritance of nucleosome state between cell cycles. The model reveals how dynamics of both nucleosome state transition and gene expression are dependent on the enzyme activities and feedback regulations. Results show that the combination of stochastic histone modification at each cell division and the deterministic feedback regulation work together to adjust the dynamics of chromatin state transition in stem cell regenerations.
Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg's sweet tooth and blocking acid export as an anticancer strategy.

PubMed

Pivovarova, Aleksandra I; MacGregor, Gordon G

2018-02-01

This study aims to investigate the utilization of The Warburg Effect, cancer's "sweet tooth" and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 μM) and the NHE1 inhibitor cariporide (10 μM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans

PubMed Central

Sierra, Crystal S.; Haase, Steven B.

2016-01-01

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation. PMID:27918582
THAP5 is a human cardiac-specific inhibitor of cell cycle that is cleaved by the proapoptotic Omi/HtrA2 protease during cell death.

PubMed

Balakrishnan, Meenakshi P; Cilenti, Lucia; Mashak, Zineb; Popat, Paiyal; Alnemri, Emad S; Zervos, Antonis S

2009-08-01

Omi/HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes.
Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

PubMed

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-02-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60-75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G 0 /G 1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G 0 /G 1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas.
Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells

PubMed Central

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-01-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60–75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G0/G1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G0/G1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas. PMID:28356992
Helper-Dependent Adenoviral Vectors.

PubMed

Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

2011-10-29

Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology.
Helper-Dependent Adenoviral Vectors

PubMed Central

Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

2012-01-01

Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer.

PubMed

Koliopanos, Alexander; Kleeff, Jörg; Xiao, Yi; Safe, Stephen; Zimmermann, Arthur; Büchler, Markus W; Friess, Helmut

2002-09-05

The arylhydrocarbon receptor (AhR) was initially identified as a member of the adaptive metabolic and toxic response pathway to polycyclic aromatic hydrocarbons and to halogenated dibenzo-p-dioxins and dibenzofurans. In the present study, we sought to determine the functional significance of the AhR pathway in pancreatic carcinogenesis. AhR expression was analysed by Northern blotting. The exact site of AhR expression was analysed by in situ hybridization and immunohistochemistry. The effects of TCDD and four selective AhR agonists on pancreatic cancer cell lines were investigated by growth assays, apoptosis assays, and induction of the cyclin-dependent kinase inhibitor p21. There was strong AhR mRNA expression in 14 out of 15 pancreatic cancer samples, weak expression in chronic pancreatitis tissues, and faint expression in all normal pancreata. In pancreatic cancer tissues, AhR mRNA and protein expression were localized in the cytoplasm of pancreatic cancer cells. TCDD and the four AhR agonists inhibited pancreatic cancer cell growth in a dose-dependent manner, and decreased anchorage-independent cell growth. DAPI staining did not reveal nuclear fragmentation and CYP1A1 and was not induced by TCDD and AhR agonists. In contrast, TCDD and AhR agonists induced the expression of the cyclin-dependent kinase inhibitor p21. In conclusion, the relatively non-toxic AhR agonists caused growth inhibition in pancreatic cancer cells with high AhR expression levels via cell cycle arrest. In addition, almost all human pancreatic cancer tissues expressed this receptor at high levels, suggesting that these or related compounds may play a role in the therapy of pancreatic cancer in the future.
Phytochemical regulation of the tumor suppressive microRNA, miR-34a, by p53-dependent and independent responses in human breast cancer cells

PubMed Central

Hargraves, Kris G.; He, Lin; Firestone, Gary L.

2016-01-01

The tumor suppressive microRNA miR-34a is transcriptionally regulated by p53 and shown to inhibit breast cancer cell proliferation as well as being a marker of increased disease free survival. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin, extracted from the sweet wormwood plant, and artesunate, a semi-synthetic derivative of artemisinin, are phytochemicals with anti-tumorigenic properties however, little is known about the role of microRNAs in their mechanism of action. Human breast cancer cells expressing wild-type (MCF-7) or mutant p53 (T47D) were treated with a concentration range and time course of each phytochemical under conditions of cell cycle arrest as detected by flow cytometry to examine the potential connection between miR-34a expression and their anti-proliferative responses. Real-time PCR and western blot analysis of extracted RNA and total protein revealed artemsinin and artesunate increased miR-34a expression in a dose-dependent manner correlating with down-regulation of the miR-34a target gene, CDK4. I3C stimulation of miR-34a expression required functional p53, whereas, both artemisinin and artesunate up-regulated miR-34a expression regardless of p53 mutational status or in the presence of dominant negative p53. Phytochemical treatments inhibited the luciferase activity of a construct containing the wild-type 3′UTR of CDK4, but not those with a mutated miR-34a binding site, whereas, transfection of miR-34a inhibitors ablated the phytochemical mediated down-regulation of CDK4 and induction of cell cycle arrest. Our results suggest that miR-34a is an essential component of the anti-proliferative activities of I3C, artemisinin and artesunate and demonstrate that both wild-type p53 dependent and independent pathways are responsible for miR-34a induction. PMID:25789847
Anti-proliferative actions of 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jung-Jin; Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764; Zhang, Wei-Yun

2011-07-22

Highlights: {yields} 2-Decylamino-DMNQ inhibited PDGF-BB-induced VSMC proliferation in a dose-dependent manner with no apparent cytotoxicity. {yields} 2-Decylamino-DMNQ inhibited PDGF-BB-induced phosphorylation of Erk1/2 and PLC{gamma}1. {yields} 2-Decylamino-DMNQ arrested a G{sub 0}/G{sub 1} cell cycle progression in association with pRb phosphorylation and PCNA expression. {yields} Both U0126, an Erk inhibitor, and U73122, a PLC{gamma} inhibitor, arrested a G{sub 0}/G{sub 1} phase of the cell cycle. -- Abstract: Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferationmore » and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-R{beta} or Akt, it did inhibit the phosphorylation of Erk1/2 and PLC{gamma}1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G{sub 0}/G{sub 1} phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLC{gamma} inhibitor, increased the proportion of cells in the G{sub 0}/G{sub 1} phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G{sub 0}/G{sub 1} phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.« less
Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

PubMed

Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

2011-01-01

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.
Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation.

PubMed

Liu, Tai-Hang; Wu, Yun-Fei; Dong, Xiao-Long; Pan, Cai-Xia; Du, Guo-Yu; Yang, Ji-Gui; Wang, Wei; Bao, Xi-Yan; Chen, Peng; Pan, Min-Hui; Lu, Cheng

2017-05-03

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.
Enterolactone induces G1-phase cell cycle arrest in non-small cell lung cancer cells by down-regulating cyclins and cyclin-dependent kinases

PubMed Central

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M.

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG) which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anti-cancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study we investigated the anti-cancer effects of EL for several non-small cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The anti-proliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL- decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1-phase. The results suggest that EL inhibits the growth of NSCLC cell lines by down-regulating G1-phase cyclins and CDKs, and up-regulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy. PMID:28323486
Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases.

PubMed

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G 1 -phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G 1 -phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21 WAF1/CIP1 , a negative regulator of the G 1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G 1 -phase cyclins and CDKs, and upregulating p21 WAF1/CIP1 , which leads to G 1 -phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.
Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Li-Wen; Hsieh, Bau-Shan; Cheng, Hsiao-Ling

2012-01-15

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growthmore » and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M phase arrest, followed by apoptosis.« less
Trichostatin A-induced apoptosis is mediated by Kruppel-like factor 4 in ovarian and lung cancer.

PubMed

Zohre, Sadeghi; Kazem, Nejati-Koshki; Abolfazl, Akbarzadeh; Mohammad, Rahmati-Yamchi; Aliakbar, Movassaghpour; Effat, Alizadeh; Zahra, Davoudi; Hassan, Dariushnejad; Nosratollah, Zarghami

2014-01-01

The istone deacetylase (HDAC) inhibitor trichostatin A (TSA) is known to mediate the regulation of gene expression and anti proliferation activity in cancer cells. Kruppel-like factor 4 (klf4) is a zinc finger- containing transcription factor of the SP/KLF family, that is expressed in a variety of tissues and regulates cell proliferation, differentiation, tumorigenesis, and apoptosis. It may either either function as a tumor suppressor or an oncogene depending on genetic context of tumors. In this study, we tested the possibility that TSA may increase klf4 expression and cancer cell growth inhibition and apoptosis in SKOV-3 and A549 cells. The cytotoxicity of TSA was determined using the MTT assay test, while klf4 gene expression was assessed by real time PCR and to ability of TSA to induce apoptosis using a Vybrant Apoptosis Assay kit. Our results showed that TSA exerted dose and time dependent cytotoxicity effect on SKOV-3 and A549 cells. Moreover TSA up-regulated klf4 expression. Flow cytometric analysis demonstrated that apoptosis was increased after TSA treatment. Taken together, this study showed that TSA increased klf4 expression in SKOV3 and A549 cell lines, consequently, klf4 may played a tumor-suppressor role by increasing both cell growth inhibition and apoptosis. This study sheds light on the details of molecular mechanisms of HDACI-induced cell cycle arrest and apoptosis.
Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less
Synchronous and Time-Dependent Expression of Cyclins, Cyclin-Dependant Kinases, and Apoptotic Genes in the Rumen Epithelia of Butyrate-Infused Goats

PubMed Central

Soomro, Jamila; Lu, Zhongyan; Gui, Hongbing; Zhang, Bei; Shen, Zanming

2018-01-01

In our previous study, we demonstrated that butyrate induced ruminal epithelial growth through cyclin D1 upregulation. Here, we investigated the influence of butyrate on the expression of genes associated with cell cycle and apoptosis in rumen epithelium. Goats (n = 24) were given an intra ruminal infusion of sodium butyrate at 0.3 (group B, n = 12) or 0 (group A, n = 12) g/kg of body weight (BW) per day before morning feeding for 28 days and were slaughtered (4 goat/group) at 5,7 and 9 h after butyrate infusion. Rumen fluid was analyzed for short chain fatty acids (SCFAs) concentration. Ruminal tissues were analyzed for morpho-histrometry and the expressions of genes associated with cell cycle and apoptosis. The results revealed that the ruminal butyrate concentration increased (P < 0.05) in B compared to group A. Morphometric analysis showed increased (P < 0.05) papillae size associated with higher number of cell layers in epithelial strata in B compared to A. Butyrate-induced papillae enlargement was coupled with enhanced mRNA expression levels (P < 0.05) of cyclin D1, CDK2, CDK4, and CDK6 (G0/G1 phase regulators) at 5 h, cyclin E1 (G1/S phase regulator) at 7 h and cyclin A and CDK1 (S phase regulators) at 9 h post-infusion compared to A group. In addition, the mRNA expression levels of apoptotic genes, i.e., caspase 3, caspase 9 and Bax at 5 h post-infusion were upregulated (P < 0.05) in group B compared to group A. The present study demonstrated that butyrate improved ruminal epithelial growth through concurrent and time-dependent changes in the expressions of genes involved in cell proliferation and apoptosis. It seems that the rate of proliferation was higher than the apoptosis which was reflected in epithelial growth. PMID:29875672
Stimulatory effect of vascular endothelial growth factor on progesterone production and survivability of cultured bubaline luteal cells.

PubMed

Chouhan, V S; Dangi, S S; Gupta, M; Babitha, V; Khan, F A; Panda, R P; Yadav, V P; Singh, G; Sarkar, M

2014-08-01

The objectives of the present study were to investigate the effects of vascular endothelial growth factor (VEGF) on progesterone (P4) synthesis in cultured luteal cells from different stages of the estrous cycle and on expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side chain cleavage (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B), antiapoptotic gene PCNA, and proapoptotic gene BAX in luteal cells obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Corpus luteum samples from the early luteal phase (ELP; day 1st-4th; n=4), MLP (day 5th-10th; n=4), and the late luteal phase (LLP; day 11th-16th; n=4) of oestrous cycle were obtained from a slaughterhouse. Luteal cell cultures were treated with VEGF (0, 1, 10 and 100 ng/ml) for 24, 48 and 72h. Progesterone was assessed by RIA, while mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Results indicated a dose- and time-dependent stimulatory effect of VEGF on P4 synthesis and expression of steroidogenic enzymes. Moreover, VEGF treatment led to an increase in PCNA expression and decrease in BAX expression. In summary, these findings suggest that VEGF acts locally in the bubaline CL to modulate steroid hormone synthesis and cell survivability, which indicates that this factor has an important role as a regulator of CL development and function in buffalo. Copyright © 2014 Elsevier B.V. All rights reserved.
Veterinary drug, 17β-trenbolone promotes the proliferation of human prostate cancer cell line through the Akt/AR signaling pathway.

PubMed

Lee, Hee-Seok; Jung, Da-Woon; Han, Songyi; Kang, Hui-Seung; Suh, Jin-Hyang; Oh, Hyun-Suk; Hwang, Myung-Sil; Moon, Guiim; Park, Yooheon; Hong, Jin-Hwan; Koo, Yong Eui

2018-05-01

Trenbolone acetate (TBA) is a synthetic anabolic steroidal growth factor that is used for rapid muscle development in cattle. The absorbed TBA is hydrolyzed to the active form, 17β-trenbolone (17 TB; 17β-hydroxy-estra-4,9,11-trien-3-one) in meat and milk products, which can cause adverse health effects in humans. Similar to 5α-dihydrotestosterone (DHT), 17 TB was reported to exhibit endocrine disrupting effects on animals and humans due to its androgenic effect via binding to the androgen receptor. The purpose of this study is to investigate the molecular mechanism of cell proliferation in prostate cancer (PCa) cells treated with 17 TB. We found that 17 TB induces AR-dependent cell proliferation in the human prostate cancer cell line, 22Rv1 in a concentration dependent manner. Treatment with 17 TB increased the expression of cell cycle regulatory proteins, cyclin D2/CDK-4 and cyclin E/CDK-2, whereas the expression of p27 was down-regulated. Furthermore, phosphorylation of Rb and activation of E2F were also induced, which suggests the activation of cyclin D2/CDK-4 and cyclin E/CDK-2 in the cells. When 22Rv1 cells were exposed to 30 pM of 17 TB, which is the effective concentration (EC 50 ) value required to observe proliferative effects on 22Rv1 cells, the expression levels of the phosphorylated forms of Akt and GSK3β were increased. This study demonstrates that 17 TB induces AR-dependent proliferation through the modulation of cell cycle-related proteins in the Akt signaling pathway. The present study provides an effective methodology for identifying cell proliferation signaling of veterinary drugs that exert AR agonistic effects. Copyright © 2018 Elsevier Ltd. All rights reserved.
Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

PubMed

Bass, Casie S; Redmer, Dale A; Kaminski, Samantha L; Grazul-Bilska, Anna T

2017-03-01

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase β protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep. © 2017 Society for Reproduction and Fertility.
MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer

PubMed Central

Ge, Xin; Jiang, Cheng-Fei; Shi, Zhu-Mei; Li, Dong-Mei; Liu, Wei-Tao; Yu, Xiaobo; Shu, Yong-Qian

2016-01-01

Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. In this study, we explored miR-137's role in the chemosensitivity of lung cancer. We found that the expression level of miR-137 is down-regulated in the human lung cancer tissues and the resistant cells strains: A549/paclitaxel(A549/PTX) and A549/cisplatin (A549/CDDP) when compared with lung cancer A549 cells. Moreover, we found that overe-expression of miR-137 inhibited cell proliferation, migration, cell survival and arrest the cell cycle in G1 phase in A549/PTX and A549/CDDP. Furthermore, Repression of miR-137 significantly promoted cell growth, migration, cell survival and cell cycle G1/S transition in A549 cells. We further demonstrated that the tumor suppressive role of miR-137 was mediated by negatively regulating Nuclear casein kinase and cyclin-dependent kinase substrate1(NUCKS1) protein expression. Importantly, miR-137 inhibits A549/PTX, A549/CDDP growth and angiogenesis in vivo. Our study is the first to identify the tumor suppressive role of over-expressed miR-137 in chemosensitivity. Identification of a novel miRNA-mediated pathway that regulates chemosensitivity in lung cancer will facilitate the development of novel therapeutic strategies in the future. PMID:26989074
The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells

PubMed Central

Brodie, Shlomit; Lee, Hae Kyung; Jiang, Wei; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M; Datta, Indrani; Kalkanis, Steve

2017-01-01

Despite advances in novel therapeutic approaches for the treatment of glioblastoma (GBM), the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need to identify molecular mechanisms that play a role in patient survival. Here, we analyzed the expression and functions of a novel lncRNA, TALNEC2 that was identified using RNA seq of E2F1-regulated lncRNAs. TALNEC2 was localized to the cytosol and its expression was E2F1-regulated and cell-cycle dependent. TALNEC2 was highly expressed in GBM with poor prognosis, in GBM specimens derived from short-term survivors and in glioma cells and glioma stem cells (GSCs). Silencing of TALNEC2 inhibited cell proliferation and arrested the cells in the G1\\S phase of the cell cycle in various cancer cell lines. In addition, silencing of TALNEC2 decreased the self-renewal and mesenchymal transformation of GSCs, increased sensitivity of these cells to radiation and prolonged survival of mice bearing GSC-derived xenografts. Using miRNA array analysis, we identified specific miRNAs that were altered in the silenced cells that were associated with cell-cycle progression, proliferation and mesenchymal transformation. Two of the downregulated miRNAs, miR-21 and miR-191, mediated some of TALNEC2 effects on the stemness and mesenchymal transformation of GSCs. In conclusion, we identified a novel E2F1-regulated lncRNA that is highly expressed in GBM and in tumors from patients of short-term survival. The expression of TALNEC2 is associated with the increased tumorigenic potential of GSCs and their resistance to radiation. We conclude that TALNEC2 is an attractive therapeutic target for the treatment of GBM. PMID:28423669
[18β-glycyrrhetinic acid piperazine derivative A30 inhibits the proliferation of SMMC-7721 hepatoma cells].

PubMed

Zhong, Like

2017-09-01

Objective To investigate the mechanism of 18β-glycyrrhetinic acid (GA) piperazine derivative A30 on the antiproliferation of hepatocellular carcinoma SMMC-7721 cells in vitro. Methods The experiment included three groups: control group, 18β-GA group and A30 group. The proliferation activity was detected by MTT assay. Cell apoptosis and the change in the cycle of SMMC-7721 cells were evaluated by flow cytometry. Western blotting was used to observe the expressions of Bcl2 and caspase-8. Results The proliferation of SMMC-7721 cells was inhibited by A30 at the concentration of 2-128 μg/mL in a dose-dependent manner. 18β-GA and A30 could induce the apoptosis of SMMC-7721 cells, and the apoptosis rate of A30 group was significantly higher than that of the 18β-GA group. In the cell cycle analysis, the G2/M phase cells of 18β-GA and A30 groups increased remarkably as compared with the control group. A30 and 18β-GA could significantly enhance the expression of caspase-8, and decreased the expression of Bcl2. Conclusion The 18β-GA piperazine derivative A30 can inhibit the proliferation of SMMC-7721 cells in vitro, and the inhibitory effect is stronger than that of 18β-GA. The mechanism may be related to the inhibition of intracellular Bcl2 protein expression and the enhancement of caspase-8 expression.
Gene expression profiling: cell cycle deregulation and aneuploidy do not cause breast cancer formation in WAP-SVT/t transgenic animals.

PubMed

Klein, Andreas; Guhl, Eva; Zollinger, Raphael; Tzeng, Yin-Jeh; Wessel, Ralf; Hummel, Michael; Graessmann, Monika; Graessmann, Adolf

2005-05-01

Microarray studies revealed that as a first hit the SV40 T/t antigen causes deregulation of 462 genes in mammary gland cells (ME cells) of WAP-SVT/t transgenic animals. The majority of deregulated genes are cell proliferation specific and Rb-E2F dependent, causing ME cell proliferation and gland hyperplasia but not breast cancer formation. In the breast tumor cells a further 207 genes are differentially expressed, most of them belonging to the cell communication category. In tissue culture breast tumor cells frequently switch off WAP-SVT/t transgene expression and regain the morphology and growth characteristics of normal ME cells, although the tumor-revertant cells are aneuploid and only 114 genes regain the expression level of normal ME cells. The profile of retransformants shows that only 38 deregulated genes are tumor-specific, and that none of them is considered to be a typical breast cancer gene.
Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S2 cells through reactive oxygen species and the mitochondria-dependent pathways.

PubMed

Huang, S-H; Hsu, M-H; Hsu, S-C; Yang, J-S; Huang, W-W; Huang, A-C; Hsiao, Y-P; Yu, C-C; Chung, J-G

2014-03-01

We have reported previously that phenethyl isothiocyanate (PEITC) induces apoptosis in human osteosarcoma U-2 OS cells. Cytotoxic activity of PEITC towards other cancer cells such as human malignant melanoma and skin cancer cells has not been reported. In this study, the anticancer activity of PEITC towards human malignant melanoma cancer A375.S2 cells was investigated. To determine the mechanisms of PEITC inhibition of cell growth, the following end points were determined in A375.S2 cells: cell morphological changes, cell cycle arrest, DNA damage and fragmentation assays and morphological assessment of nuclear change, reactive oxygen species (ROS) and Ca(2+) generations, mitochondrial membrane potential disruption, and nitric oxide and 10-N-nonyl acridine orange productions, expression and activation of caspase-3 and -9, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, poly (adenosine diphosphate-ribose) polymerase, and cytochrome c release, apoptosis-inducing factor and endonuclease G. PEITC induced morphological changes in time- and dose-dependent manner. PEITC induced G2/M phase arrest and induced apoptosis via endoplasmic reticulum stress-mediated mitochondria-dependent pathway. Western blot analysis showed that PEITC promoted Bax expression and inhibited Bcl-2 expression associated with the disintegration of the outer mitochondrial membrane causing cytochrome c release, and activation of caspase-9 and -3 cascade leading to apoptosis. We conclude that PEITC-triggered apoptotic death in A375.S2 cells occurs through ROS-mediated mitochondria-dependent pathways.
Effects of sodium phenylbutyrate on differentiation and induction of the P21WAF1/CIP1 anti-oncogene in human liver carcinoma cell lines.

PubMed

Meng, Mei; Jiang, Jun Mei; Liu, Hui; In, Cheng Yong; Zhu, Ju Ren

2005-01-01

To explore the effects of sodium phenylbutyrate on the proliferation, differentiation, cell cycle arrest and induction of the P(21WAF1/CIP1) anti-oncogene in human liver carcinoma cell lines Bel-7402 and HepG2. Bel-7402 and HepG2 human liver carcinoma cells were treated with sodium phenylbutyrate at different concentrations. Light microscopy was used to observe morphological changes in the carcinoma cells. Effects on the cell cycle were detected by using flow cytometry. P(21WAF1/CIP1) expression was determined by both reverse transcription-polymerase chain reaction and western blotting. Statistical analysis was performed by using one-way anova and Student's t-test. Sodium phenylbutyrate treatment caused time- and dose-dependent growth inhibition of Bel-7402 and HepG2 cells. This treatment also caused a decline in the proportion of S-phase cells and an increase in the proportion of G(0)/G(1) cells. Sodium phenylbutyrate increased the expression of P(21WAF1/CIP1). Sodium phenylbutyrate inhibits the proliferation of human liver carcinoma cells Bel-7402 and HepG2, induces partial differentiation, and increases the expression of P(21WAF1/CIP1).

Versatile function of the circadian protein CIPC as a regulator of Erk activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsunaga, Ryota; Nishino, Tasuku; Yokoyama, Atsushi

2016-01-15

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase,more » and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.« less
Berberine and Evodiamine Act Synergistically Against Human Breast Cancer MCF-7 Cells by Inducing Cell Cycle Arrest and Apoptosis.

PubMed

Du, Jia; Sun, Yang; Lu, Yi-Yu; Lau, Eric; Zhao, Ming; Zhou, Qian-Mei; Su, Shi-Bing

2017-11-01

The synergistic combinations of natural products have long been the basis of Traditional Chinese herbal Medicine formulas. In this study, we investigated the synergistic effects of a combination of berberine and evodiamine against human breast cancer MCF-7 cells in vitro and in vivo, and explored its mechanism. Cell survival was measured using the MTT assay. Apoptosis-related proteins were observed using western blot analysis. Apoptosis was detected with flow cytometric analysis and by Hoechst 33258 staining. Tumor xenografts were used in vivo. Compared to berberine or evodiamine treatments alone, the combination treatment of berberine (25 μM) and evodiamine (15 μM) synergistically inhibited the proliferation of MCF-7 cells in a time-dependent manner and resulted in the G 0 /G 1 phase accumulation of cells that exhibited increased expression levels of the CDK inhibitors p21 and p27 with a concomitant reduction in the expression levels of cell-cycle checkpoint proteins cyclin D1, cyclin E, CDK4, and CDK6. Furthermore, the combination treatment induced apoptosis that was accompanied by increased expression levels of p53 and Bax, reduced expression levels of Bcl-2, activation of caspase-7, and caspase-9, and the cleavage of PARP. The combination of berberine and evodiamine synergistically inhibited tumor growth in vivo in MCF-7 human breast cancer xenografts. Combination of berberine and evodiamine acts synergistically to suppress the proliferation of MCF-7 cells by inducing cell cycle arrest and apoptosis, illustrating the potential synergistic and combinatorial application of bioactive natural products. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.

2000-01-01

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems. PMID:10648628
Protoparvovirus Interactions with the Cellular DNA Damage Response

PubMed Central

Majumder, Kinjal; Etingov, Igor

2017-01-01

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection. PMID:29088070
Protoparvovirus Interactions with the Cellular DNA Damage Response.

PubMed

Majumder, Kinjal; Etingov, Igor; Pintel, David J

2017-10-31

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.
DEC1 regulates breast cancer cell proliferation by stabilizing cyclin E protein and delays the progression of cell cycle S phase

PubMed Central

Bi, H; Li, S; Qu, X; Wang, M; Bai, X; Xu, Z; Ao, X; Jia, Z; Jiang, X; Yang, Y; Wu, H

2015-01-01

Breast cancer that is accompanied by a high level of cyclin E expression usually exhibits poor prognosis and clinical outcome. Several factors are known to regulate the level of cyclin E during the cell cycle progression. The transcription factor DEC1 (also known as STRA13 and SHARP2) plays an important role in cell proliferation and apoptosis. Nevertheless, the mechanism of its role in cell proliferation is poorly understood. In this study, using the breast cancer cell lines MCF-7 and T47D, we showed that DEC1 could inhibit the cell cycle progression of breast cancer cells independently of its transcriptional activity. The cell cycle-dependent timing of DEC1 overexpression could affect the progression of the cell cycle through regulating the level of cyclin E protein. DEC1 stabilized cyclin E at the protein level by interacting with cyclin E. Overexpression of DEC1 repressed the interaction between cyclin E and its E3 ligase Fbw7α, consequently reducing the level of polyunbiquitinated cyclin E and increased the accumulation of non-ubiquitinated cyclin E. Furthermore, DEC1 also promoted the nuclear accumulation of Cdk2 and the formation of cyclin E/Cdk2 complex, as well as upregulating the activity of the cyclin E/Cdk2 complex, which inhibited the subsequent association of cyclin A with Cdk2. This had the effect of prolonging the S phase and suppressing the growth of breast cancers in a mouse xenograft model. These events probably constitute the essential steps in DEC1-regulated cell proliferation, thus opening up the possibility of a protein-based molecular strategy for eliminating cancer cells that manifest a high-level expression of cyclin E. PMID:26402517
DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53more » status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.« less
Role of melatonin in the epigenetic regulation of breast cancer.

PubMed

Korkmaz, Ahmet; Sanchez-Barcelo, Emilio J; Tan, Dun-Xian; Reiter, Russel J

2009-05-01

The oncostatic properties of melatonin as they directly or indirectly involve epigenetic mechanisms of cancer are reviewed with a special focus on breast cancer. Five lines of evidence suggest that melatonin works via epigenetic processes: (1) melatonin influences transcriptional activity of nuclear receptors (ERalpha, GR and RAR) involved in the regulation of breast cancer cell growth; (2) melatonin down-regulates the expression of genes responsible for the local synthesis or activation of estrogens including aromatase, an effect which may be mediated by methylation of the CYP19 gene or deacetylation of CYP19 histones; (3) melatonin inhibits telomerase activity and expression induced by either natural estrogens or xenoestrogens; (4) melatonin modulates the cell cycle through the inhibition of cyclin D1 expression; (5) melatonin influences circadian rhythm disturbances dependent on alterations of the light/dark cycle (i.e., light at night) with the subsequent deregulation of PER2 which acts as a tumor suppressor gene.
The role of p21Waf1/CIP1 as a Cip/Kip type cell-cycle regulator in oral squamous cell carcinoma (Review).

PubMed

Pérez-Sayáns, Mario; Suárez-Peñaranda, José-Manuel; Gayoso-Diz, Pilar; Barros-Angueira, Francisco; Gándara-Rey, José-Manuel; García-García, Abel

2013-03-01

Oral Squamous Cell Carcinoma (OSCC) is biologically characterized by the accumulation of multiple genetic and molecular alterations that end up clinically characterized as a malignant neoplasm through a phenomenon known as multistep. The members of the Cip/Kip family, specifically p21Waf1/CIP1, are responsible for cell cycle control, blocking the transition from phase G1 to phase S. We made a search of articles of peer-reviewed Journals in PubMed/ Medline, crossing the keywords. The goal of this paper is to determine the relationship between p21Waf1/CIP1 expression and several clinical and pathological aspects of OSCC, their relationship with p53 and HPV, as well as genetic alterations in their expression pattern, their use as a prognosis market in the evolution of precancerous lesions and their roles in anticancer treatments. The results of p21WAF1/CIP1 expression in OSCC showed mixed results in terms of positivity/negativity throughout different studies. It seems that, although p21Waf1/CIP1 expression is controlled in a p53-dependent manner, coexpression of both in OSCC is not intrinsically related. Although the presence of HPV viral oncoproteins increases p21Waf1/CIP1 levels, the small number of studies, have forced us to disregard the hypothesis that HPV infected lesions that present better prognosis are due to a p21Waf1/CIP1-dependent control. The role of p21WAF1/CIP1 as cell-cycle regulator has been well described; however, its relationship to OSCC, the clinical and pathological variables of tumors, HPV and different treatments are not entirely clear. Thus, it would be very interesting to pursue further study of this protein, which may have a significant value for the diagnosis, prognosis and therapy of this type of tumors.
Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

PubMed Central

Lammers, Twan; Peschke, Peter; Ehemann, Volker; Debus, Jürgen; Slobodin, Boris; Lavi, Sara; Huber, Peter

2007-01-01

Background PP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis. Results It was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased. Conclusion Based on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer. PMID:17941990
Disruption of the G1/S Transition in Human Papillomavirus Type 16 E7-Expressing Human Cells Is Associated with Altered Regulation of Cyclin E

PubMed Central

Martin, Larry G.; Demers, G. William; Galloway, Denise A.

1998-01-01

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F. PMID:9444990
Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

PubMed

Kraljević Pavelić, Sandra; Marjanović, Marko; Poznić, Miroslav; Kralj, Marijeta

2009-12-01

p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.
MINA controls proliferation and tumorigenesis of glioblastoma by epigenetically regulating cyclins and CDKs via H3K9me3 demethylation.

PubMed

Huang, M-Y; Xuan, F; Liu, W; Cui, H-J

2017-01-19

It is generally known that histone demethylases regulate gene transcription by altering the methylate status on histones, but their roles in cancers and the underlying molecular mechanisms still remain unclear. MYC-induced nuclear antigen (MINA) is reported to be a histone demethylase and highly expressed in many cancers. Here, for the first time, we show that MINA is involved in glioblastoma carcinogenesis and reveal the probable mechanisms of it in cell-cycle control. Kaplan-Meier analysis of progression-free survival showed that high MINA expression was strongly correlated with poor outcome and advancing tumor stage. MINA knockdown significantly repressed the cell proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo that were rescued by overexpressing the full-length MINA afterwards. Microarray analysis after knockdown of MINA revealed that MINA probably regulated glioblastoma carcinogenesis through the predominant cell-cycle pathways. Further investigation showed that MINA deficiency led to a cell-cycle arrest in G1 and G2 phases. And among the downstream genes, we found that cyclins and cyclin-dependent kinases were directly activated by MINA via the demethylation of H3K9me3.
Regulation of Androgen Receptor Expression Alters AMPK Phosphorylation in the Endometrium: In Vivo and In Vitro Studies in Women with Polycystic Ovary Syndrome

PubMed Central

Li, Xin; Pishdari, Bano; Cui, Peng; Hu, Min; Yang, Hong-Ping; Guo, Yan-Rong; Jiang, Hong-Yuan; Feng, Yi; Billig, Håkan; Shao, Ruijin

2015-01-01

The failure of reproductive success in polycystic ovary syndrome (PCOS) patients could be in part due to endometrial dysfunction. However, no studies have investigated any causality between androgen, androgen receptor (AR) expression, and adenosine monophosphate activated protein kinase (AMPK) activation in the endometrium under physiological and pathological conditions. In the present study, we show that 1) endometrial AR expression levels fluctuate in non-PCOS and PCOS patients during the menstrual cycle; 2) the menstrual phase-dependent alteration of p-AMPKα expression occurs in non-PCOS patients but not in PCOS patients; 3) AR expression is higher in PCOS patients than non-PCOS patients during hyperplasia while AMPKα activation (indicated by the ratio of p-AMPKα to AMPKα); and 4) co-localization of AR and Ki-67 in epithelial cell nuclei is observed in endometrial hyperplasia. Importantly, using in vitro human tissue culture and an in vivo 5α-dihydrotestosterone-treated rat model, we show that the action of androgen on AMPKα activation is likely mediated through nuclear AR, especially in epithelial cells. Collectively, we present evidence that AR expression and AMPKα activation depend on menstrual cycle phase and the presence of PCOS, and the data suggest that AR-mediated regulation of AMPKα activation might play a role in the development of endometrial hyperplasia. PMID:26681917
Human transcription factor hTAF(II)150 (CIF150) is involved in transcriptional regulation of cell cycle progression.

PubMed

Martin, J; Halenbeck, R; Kaufmann, J

1999-08-01

Here we present evidence that CIF150 (hTAF(II)150), the human homolog of Drosophila TAF(II)150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAF(II)150) seems to be less tightly associated with human transcription factor IID than hTAF(II)130 is associated with hTAF(II)250. The transient functional knockout of CIF150 (hTAF(II)150) protein led to cell cycle arrest at the G(2)/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAF(II)150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAF(II)150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAF(II)150) function. We defined a CIF150 (hTAF(II)150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAF(II)150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression.
Human Transcription Factor hTAFII150 (CIF150) Is Involved in Transcriptional Regulation of Cell Cycle Progression

PubMed Central

Martin, Jay; Halenbeck, Robert; Kaufmann, Jörg

1999-01-01

Here we present evidence that CIF150 (hTAFII150), the human homolog of Drosophila TAFII150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAFII150) seems to be less tightly associated with human transcription factor IID than hTAFII130 is associated with hTAFII250. The transient functional knockout of CIF150 (hTAFII150) protein led to cell cycle arrest at the G2/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAFII150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAFII150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAFII150) function. We defined a CIF150 (hTAFII150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAFII150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression. PMID:10409744
3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells.

PubMed

Zuo, Daiying; Guo, Dandan; Jiang, Xuewei; Guan, Qi; Qi, Huan; Xu, Jingwen; Li, Zengqiang; Yang, Fushan; Zhang, Weige; Wu, Yingliang

2015-02-05

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
The cytotoxic activity of Ziziphus Jujube on cervical cancer cells: In Vitro study.

PubMed

Hoshyar, R; Jamali, S; Fereidouni, M; Abedini, M R

2015-12-30

Recently, there are tendency to use natural products such as Ziziphus Jujube (Jujube) as therapeutic agents for cancer. Understanding the molecular mechanisms of anti-cancer effects of Jujube may improve the current therapeutic strategies against cervical cancer. Our MTT data showed a significant dose- and time-dependent inhibition of OV-2008 cell proliferation following Jujube administration. Moreover, qRT-PCR analyses significantly revealed the suppression of cyclin D1 and the enhancement of P53, P21 and P27 expression in treated cells. These results suggest that the herb exerts a cytotoxic effect on cervical cancer cells through alternation of the expression of the genes that are involved in regulation of cell cycle.
DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

PubMed Central

Jagot-Lacoussiere, Léonard; Faye, Audrey; Bruzzoni-Giovanelli, Heriberto; Villoutreix, Bruno O; Rain, Jean-Christophe; Poyet, Jean-Luc

2015-01-01

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response. PMID:25695197
Curcumin induces apoptosis in human leukemic cell lines through an IFIT2-dependent pathway

PubMed Central

Zhang, Yonglu; Kong, Yunyuan; Liu, Shuyuan; Zeng, Lingbing; Wan, Lagen; Zhang, Zhanglin

2017-01-01

ABSTRACT Curcumin, the primary bioactive component isolated from turmeric, has been shown to possess variety of biologic functions including anti-cancer activity. However, molecular mechanisms in different cancer cells are various. In the present study, we demonstrated that curcumin induced G2/M cell cycle arrest and apoptosis by increasing the expression levels of cleaved caspase-3, cleaved PARP and decreasing the expression of BCL−2 in U937 human leukemic cells but not in K562 cells. We found some interferon induced genes, especially interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were significantly upregulated when treated with curcumin in U937 cells by gene expression chip array, and further confirmed that the expression of IFIT2 was obviously higher in U937 than that in K562 cells by Western blot assay. In addition, inhibiting the expression of IFIT2 by shRNA in U937 rescued curcumin-induced apoptosis and exogenous overexpression of IFIT2 by lentiviral transduction or treating with IFNγ in K562 cells enhanced anti-cancer activity of curcumin. These results indicated for the first time that curcumin induced leukemic cell apoptosis via an IFIT2-dependent signaling pathways. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for curcumin combined with interferon in the cancer therapeutics. PMID:28071969

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shim, Ki Shuk; Department of Neonatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna; Rosner, Margit

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could representmore » a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.« less
HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

PubMed

Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

2015-10-27

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.
A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle.

PubMed

Linke, Christian; Chasapi, Anastasia; González-Novo, Alberto; Al Sawad, Istabrak; Tognetti, Silvia; Klipp, Edda; Loog, Mart; Krobitsch, Sylvia; Posas, Francesc; Xenarios, Ioannis; Barberis, Matteo

2017-01-01

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.
The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

PubMed

Shirazi Fard, Shahrzad; Thyselius, Malin; All-Ericsson, Charlotta; Hallböök, Finn

2014-01-01

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.
Alteration of cell cycle progression by Sindbis virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Veromore » cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.« less
PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage

PubMed Central

Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

2012-01-01

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments. PMID:23235459
PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage.

PubMed

Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

2012-12-13

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments.
Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

PubMed Central

Peng, Xu; Karuturi, R. Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

2005-01-01

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC. PMID:15616197
Pre-exposure to 50 Hz-electromagnetic fields enhanced the antiproliferative efficacy of 5-fluorouracil in breast cancer MCF-7 cells

PubMed Central

Chen, Sha; Sun, Xiongshan; Guan, Xiao; Yang, Yao; Peng, Bingjie; Pan, Xiaodong; Li, Jinfang; Yi, Weijing; Li, Peng; Zhang, Hongwei; Feng, Dongfang; Chen, An; Li, Xiaohui; Yin, Zuoming

2018-01-01

Resistance to 5-fluorouracil (5-FU) and its induced immune suppression have prevented its extensive application in the clinical treatment of breast cancer. In this study, the combined effect of 50 Hz-EMFs and 5-FU in the treatment of breast cancer was explored. MCF-7 and MCF10A cells were pre-exposed to 50 Hz-EMFs for 0, 2, 4, 8 and 12 h and then treated with different concentrations of 5-FU for 24 h; cell viability was analyzed by MTT assay and flow cytometry. After pre-exposure to 50 Hz-EMFs for 12 h, apoptosis and cell cycle distribution in MCF-7 and MCF10A cells were detected via flow cytometry and DNA synthesis was measured by EdU incorporation assay. Apoptosis-related and cell cycle-related gene and protein expression levels were monitored by qPCR and western blotting. Pre-exposure to 50 Hz-EMFs for 12 h enhanced the antiproliferative effect of 5-FU in breast cancer cell line MCF-7 in a dose-dependent manner but not in normal human breast epithelial cell line MCF10A. Exposure to 50 Hz-EMFs had no effect on apoptosis and P53 expression of MCF-7 and MCF10A cells, whereas it promoted DNA synthesis, induced entry of MCF-7 cells into the S phase of cell cycle, and upregulated the expression levels of cell cycle-related proteins Cyclin D1 and Cyclin E. Considering the pharmacological mechanisms of 5-FU in specifically disrupting DNA synthesis, this enhanced inhibitory effect might have resulted from the specific sensitivity of MCF7 cells in active S phase to 5-FU. Our findings demonstrate the enhanced cytotoxic activity of 5-FU on MCF7 cells through promoting entry into the S phase of the cell cycle via exposure to 50 Hz-EMFs, which provides a novel method of cancer treatment based on the combinatorial use of 50 Hz-EMFs and chemotherapy. PMID:29617363
Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblasticmore » cells.« less
Amarogentin secoiridoid inhibits in vivo cancer cell growth in xenograft mice model and induces apoptosis in human gastric cancer cells (SNU-16) through G2/M cell cycle arrest and PI3K/Akt signalling pathway.

PubMed

Zhao, Jian-Guo; Zhang, Ling; Xiang, Xiao-Jun; Yu, Feng; Ye, Wan-Li; Wu, Dong-Ping; Wang, Jian-Fang; Xiong, Jian-Ping

2016-01-01

To investigate the in vitro and in vivo antitumor effects of amarogentin in SNU-16 human gastric cancer cells as well as in nude mice xenograft model. The effects of this compound on cell apoptosis, cell cycle phase distribution and PI3K/Akt and m-TOR signalling pathways were also studied in detail. MTT assay was used to study the effect of amarogentin on SNU-16 cell viability while clonogenic assay indicated the effect of the compound on colony formation tendency of these cells. Phase contrast microscopy revealed the effect on cellular morphology while flow cytometry was engaged to study the effects on cell apoptosis and cell cycle arrest. SNU-16 cancer cells were subcutaneously inoculated into nude mice to investigate the in vivo antitumor effects of amarogentin. Amarogentin induced potent, dose-dependent as well as time-dependent cytotoxic effects on the growth of SNU-16 human gastric cancer cells. Amarogentin also inhibited the colony forming capability of these tumor cells and its treatment led to morphological alterations in these cells in which the cells became withered and rounded, detached from one another and adopted irregular shapes while floating freely in the culture medium. In comparison to untreated control cells, the amarogentin treated cells with 10, 50 and 75 μM exhibited 32.5, 45.2 and 57.1 % apoptotic cells, respectively. Amarogentin induced potent and dose-dependent G2/M cell cycle arrest in these cells and led to downregulation of m-TOR, p-PI3K, PI3K, p-Akt and Akt and upregulation of cyclin D1 and cyclin E protein expressions. The tumor tissues obtained from the amarogentin-treated mice were much smaller than the tumor tissues derived from the control group. Amarogentin exerts potent in vitro and in vivo antitumor effects in SNU-16 cell model as well as in nude mice xenograft model. These antitumor effects were found to be mediated through apoptosis induction, G2/M cell cycle arrest and downregulation of PI3K/Akt/m-TOR signalling pathways.
FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways

PubMed Central

Kelleher, Fergal C.; O'sullivan, Hazel

2016-01-01

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin – cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome. FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of β-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy. PMID:27074562
Inhibitory effect of genistein on the invasive potential of human cervical cancer cells via modulation of matrix metalloproteinase-9 and tissue inhibitors of matrix metalloproteinase-1 expression.

PubMed

Hussain, Arif; Harish, Geetganga; Prabhu, Sathyen Alwin; Mohsin, Javeria; Khan, Munawwar Ali; Rizvi, Tahir A; Sharma, Chhavi

2012-12-01

One of the most challenging stumbling blocks for the treatment of cancer is the ability of cancer cells to break the natural barriers and spread from its site of origin to non-adjacent regional and distant sites, accounting for high cancer mortality rates. Gamut experimental and epidemiological data advocate the use of pharmacological or nutritional interventions to inhibit or delay various stage(s) of cancer such as invasion and metastasis. Genistein, a promising chemopreventive agent, has gained considerable attention for its powerful anti-carcinogenic, anti-angiogenic and chemosensitizing activities. In this study, the cytotoxic potential of genistein on HeLa cells by cell viability assay and the mode of cell death induced by genistein were determined by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Moreover, to establish its inhibitory effect on migration of HeLa cells, scratch wound assay was performed and these results were correlated with the expression of genes involved in invasion and migration (MMP-9 and TIMP-1) by RT-PCR. The exposure of HeLa cells to genistein resulted in significant dose- and time-dependent growth inhibition, which was found to be mediated by apoptosis and cell cycle arrest at G(2)/M phase. In addition, it induced migration-inhibition in a time-dependent manner by modulating the expression of MMP-9 and TIMP-1. Our results signify that genistein may be an effective anti-neoplastic agent to prevent cancer cell growth and invasion and metastasis. Therefore therapeutic strategies utilizing genistein could be developed to substantially reduce cancer morbidity and mortality. Copyright © 2012 Elsevier Ltd. All rights reserved.
Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074
Effects of Histone Deacetylase Inhibitor Panobinostat (LBH589) on Bone Marrow Mononuclear Cells of Relapsed or Refractory Multiple Myeloma Patients and Its Mechanisms

PubMed Central

Ma, Yanping; Liu, Wenhua; Zhang, Ling; Jia, Gu

2017-01-01

Background The aim of this study was to explore the impact of LBH589 alone or in combination with proteasome inhibitor bortezomib on multiple myeloma (MM) cell proliferation and its mechanism. Material/Methods MM cell line U266 and RRMM-BMMNC were treated with different concentrations of LBH589 alone or in combination with bortezomib. Cell proliferation was detected by MTT assay. Cell cycle and apoptosis was analyzed by flow cytometry. The protein and mRNA level of related genes was determined by Western blotting and qRT-PCR respectively. Results U266 cell and RRMM-BMMNC proliferation were inhibited by different concentrations of LBH589 (0, 10, 20, and 50 nmol/L) alone or 50 nmol/L of LBH589 in combination with bortezomib (10 and 20 nmol/L) in a dose- and time-dependent manner. LBH589 significantly induced G0/G1phase arrest and apoptosis in RRMM-BMMNC in a dose-dependent manner. The effects were significantly higher in all combined groups than in single-agent groups (all P<0.05). The mRNA level of Caspase3 and APAF1 were up-regulated gradually, while TOSO gene expression in RRMM-BMMNC was down-regulated gradually in a dose- and time-dependent manner. Moreover, LBH589 significantly induced hyperacetylation of histone H4, the protein level of PARP notably increased, and the level of Bcl-X decreased. Conclusions LBH589 can inhibit MM cell growth, block the cell cycle, and induce cell apoptosis, which has an anti-resistant effect on multidrug-resistant cells. LBH589 in combination with bortezomib has a synergistic effect on myeloma cells; its mechanism and reversal of drug resistance mechanism is involved in multiple changes in gene expression. PMID:29080899
Intrinsic, nondeterministic circadian rhythm generation in identified mammalian neurons.

PubMed

Webb, Alexis B; Angelo, Nikhil; Huettner, James E; Herzog, Erik D

2009-09-22

Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm(2) eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.
Light-Dependent Transcriptional Regulation of Genes of Biogeochemical Interest in the Diploid and Haploid Life Cycle Stages of Emiliania huxleyi▿ †

PubMed Central

Richier, Sophie; Kerros, Marie-Emmanuelle; de Vargas, Colomban; Haramaty, Liti; Falkowski, Paul G.; Gattuso, Jean-Pierre

2009-01-01

The expression of genes of biogeochemical interest in calcifying and noncalcifying life stages of the coccolithophore Emiliania huxleyi was investigated. Transcripts potentially involved in calcification were tested through a light-dark cycle. These transcripts were more abundant in calcifying cells and were upregulated in the light. Their application as potential candidates for in situ biogeochemical proxies is also suggested. PMID:19304825
Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.

PubMed

Zhang, Ji; Fan, Jing; Venneti, Sriram; Cross, Justin R; Takagi, Toshimitsu; Bhinder, Bhavneet; Djaballah, Hakim; Kanai, Masayuki; Cheng, Emily H; Judkins, Alexander R; Pawel, Bruce; Baggs, Julie; Cherry, Sara; Rabinowitz, Joshua D; Thompson, Craig B

2014-10-23

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.
Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sumrejkanchanakij, Piyamas; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330; Eto, Kazuhiro

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, amore » process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.« less
Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

PubMed

Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression*

PubMed Central

Wang, Chunmei; Qi, Runzi; Li, Nan; Wang, Zhengxin; An, Huazhang; Zhang, Qinghua; Yu, Yizhi; Cao, Xuetao

2009-01-01

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC. PMID:19376776
Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling.

PubMed

Zhao, Li; Li, Jing; Hao, Yan Hui; Gao, Ya Bing; Wang, Shui Ming; Zhang, Jing; Dong, Ji; Zhou, Hong Mei; Liu, Shu Chen; Peng, Rui Yun

2017-05-01

To investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms. NK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01). Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.
Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Y.-J.; Department of Biotechnology, Asia University, Taichung, Taiwan; Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan

2009-01-23

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, wemore » observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI{sub 50}) concentration of 2.35 {mu}M. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.« less
miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xuesong; Gong, Xuhai; Chen, Jing

Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defectmore » in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3′UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma. - Highlights: • miR-340 is downregulated in glioblastoma samples and cell lines. • miR-340 inhibits glioblastoma cell proliferation. • miR-340 directly targets CDK6, cyclin-D1, and cyclin-D2. • miR-340 regulates glioblastoma cell proliferation via CDK6, cyclin-D1 and cyclin-D2.« less
Functional Study of the Vitamin K Cycle Enzymes in Live Cells

PubMed Central

Tie, J.-K.; Stafford, D.W.

2018-01-01

Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle. PMID:28065270
Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT

PubMed Central

Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT. PMID:29590128
Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT.

PubMed

Xu, Xiaojun; Liu, Xiaoyuan; Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT.
The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components

PubMed Central

García-Cruz, Karla V.; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A.; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R.

2016-01-01

Background Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. Methods We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. Key Results We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a. In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. Conclusion XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. PMID:27474508
Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

PubMed

Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

2010-11-01

Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.
Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

PubMed

McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

2014-01-01

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.
Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

PubMed

Delporte, Annelies; De Zaeytijd, Jeroen; De Storme, Nico; Azmi, Abdelkrim; Geelen, Danny; Smagghe, Guy; Guisez, Yves; Van Damme, Els J M

2014-10-01

The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

PubMed

Local, Andrea; Zhang, Hongying; Benbatoul, Khalid D; Folger, Peter; Sheng, Xia; Tsai, Cheng-Yu; Howell, Stephen B; Rice, William G

2018-06-01

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G 0 -G 1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253) 3 ]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253) 3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253) 3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR . ©2018 American Association for Cancer Research.
Inhibition of AMPK and Krebs cycle gene expression drives metabolic remodeling of Pten-deficient preneoplastic thyroid cells.

PubMed

Antico Arciuch, Valeria G; Russo, Marika A; Kang, Kristy S; Di Cristofano, Antonio

2013-09-01

Rapidly proliferating and neoplastically transformed cells generate the energy required to support rapid cell division by increasing glycolysis and decreasing flux through the oxidative phosphorylation (OXPHOS) pathway, usually without alterations in mitochondrial function. In contrast, little is known of the metabolic alterations, if any, which occur in cells harboring mutations that prime their neoplastic transformation. To address this question, we used a Pten-deficient mouse model to examine thyroid cells where a mild hyperplasia progresses slowly to follicular thyroid carcinoma. Using this model, we report that constitutive phosphoinositide 3-kinase (PI3K) activation caused by PTEN deficiency in nontransformed thyrocytes results in a global downregulation of Krebs cycle and OXPHOS gene expression, defective mitochondria, reduced respiration, and an enhancement in compensatory glycolysis. We found that this process does not involve any of the pathways classically associated with the Warburg effect. Moreover, this process was independent of proliferation but contributed directly to thyroid hyperplasia. Our findings define a novel metabolic switch to glycolysis driven by PI3K-dependent AMPK inactivation with a consequent repression in the expression of key metabolic transcription regulators. ©2013 AACR.
Phospho-Rb mediating cell cycle reentry induces early apoptosis following oxygen-glucose deprivation in rat cortical neurons.

PubMed

Yu, Ying; Ren, Qing-Guo; Zhang, Zhao-Hui; Zhou, Ke; Yu, Zhi-Yuan; Luo, Xiang; Wang, Wei

2012-03-01

The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured cortical neurons following oxygen-glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.
A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.

PubMed

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.
A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

PubMed Central

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001 PMID:24596151
Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481
The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

PubMed

Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

2014-01-01

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.
The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

PubMed Central

Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

2014-01-01

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285
Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress, AMPK and FOXO3a in MCF-7 Breast Cancer Cells

PubMed Central

Queiroz, Eveline A. I. F.; Puukila, Stephanie; Eichler, Rosangela; Sampaio, Sandra C.; Forsyth, Heidi L.; Lees, Simon J.; Barbosa, Aneli M.; Dekker, Robert F. H.; Fortes, Zuleica B.; Khaper, Neelam

2014-01-01

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role. PMID:24858012

Cajanol, a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway.

PubMed

Luo, Meng; Liu, Xia; Zu, Yuangang; Fu, Yujie; Zhang, Su; Yao, Liping; Efferth, Thomas

2010-10-06

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (DeltaPsim) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC(50) value was 54.05 microM after 72 h treatment, 58.32 microM after 48 h; and 83.42 microM after 24h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.
Effects of phenformin on the proliferation of human tumor cell lines.

PubMed

Caraci, Filippo; Chisari, Mariangela; Frasca, Giuseppina; Chiechio, Santina; Salomone, Salvatore; Pinto, Antonio; Sortino, Maria Angela; Bianchi, Alfredo

2003-12-19

Phenformin is a biguanide that has been largely used in the past for its anti-diabetic activity. A large body of evidence suggests additional effects of phenformin including antitumoral activity in different animal models in vivo. Thus, the present study has been conducted in order to elucidate possible mechanisms involved in the antitumoral effects of phenformin. In various tumoral cell lines (SH-SY5Y neuroblastoma and LNCaP prostate adenocarcinoma cells), increasing concentrations of phenformin (50-500 microM) induced a concentration-dependent inhibition of cell proliferation. This effect was not dependent on the ability of the drug to reduce glucose levels and was accompanied by induction of apoptotic cell death as measured by cytofluorometric analysis. In addition, a short-time incubation of SH-SY5Y cells with phenformin induced enhanced and transient expression of the cell cycle inhibitor p21 suggesting that phenformin causes inhibition of cell cycle progression prior to induction of apoptosis. These results demonstrate an activity at the cellular level of phenformin that supports its antitumoral effect observed in vivo.
Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

PubMed

Ding, Xia; He, Zhuohao; Zhou, Kechun; Cheng, Ju; Yao, Hailan; Lu, Dongliang; Cai, Rong; Jin, Yening; Dong, Bin; Xu, Yinghui; Wang, Yizheng

2010-07-21

Patients with glioblastoma multiforme, the most aggressive form of glioma, have a median survival of approximately 12 months. Calcium (Ca(2+)) signaling plays an important role in cell proliferation, and some members of the Ca(2+)-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of many types of cancer cells. In this study, we investigated the role of TRPC6 in cell cycle progression and in the development of human glioma. TRPC6 protein and mRNA expression were assessed in glioma (n = 33) and normal (n = 17) brain tissues from patients and in human glioma cell lines U251, U87, and T98G. Activation of TRPC6 channels was tested by platelet-derived growth factor-induced Ca(2+) imaging. The effect of inhibiting TRPC6 activity or expression using the dominant-negative mutant TRPC6 (DNC6) or RNA interference, respectively, was tested on cell growth, cell cycle progression, radiosensitization of glioma cells, and development of xenografted human gliomas in a mouse model. The green fluorescent protein (GFP) and wild-type TRPC6 (WTC6) were used as controls. Survival of mice bearing xenografted tumors in the GFP, DNC6, and WTC6 groups (n = 13, 15, and 13, respectively) was compared using Kaplan-Meier analysis. All statistical tests were two-sided. Functional TRPC6 was overexpressed in human glioma cells. Inhibition of TRPC6 activity or expression attenuated the increase in intracellular Ca(2+) by platelet-derived growth factor, suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 activation and cell division cycle 25 homolog C expression regulated the cell cycle arrest. Inhibition of TRPC6 activity also reduced tumor volume in a subcutaneous mouse model of xenografted human tumors (P = .014 vs GFP; P < .001 vs WTC6) and increased mean survival in mice in an intracranial model (P < .001 vs GFP or WTC6). In this preclinical model, TRPC6 channels were essential for glioma development via regulation of G2/M phase transition. This study suggests that TRPC6 might be a new target for therapeutic intervention of human glioma.
Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

PubMed

Guo, Lei; Xiao, Yongsheng; Fan, Ming; Li, Jian Jian; Wang, Yinsheng

2015-01-02

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.
Characterization of the Regulation and Function of Zinc-Dependent Histone Deacetylases During Mouse Liver Regeneration

PubMed Central

Huang, Jiansheng; Barr, Emily; Rudnick, David A.

2013-01-01

The studies reported here were undertaken to define the regulation and functional importance of zinc-dependent histone deacetylase (Zn-HDAC) activity during liver regeneration using the mouse partial hepatectomy (PH) model. The results showed that hepatic HDAC activity was significantly increased in nuclear and cytoplasmic fractions following PH. Further analyses showed isoform-specific effects of PH on HDAC mRNA and protein expression, with increased expression of the class I HDACs, 1 and 8, and class II HDAC4 in regenerating liver. Hepatic expression of (class II) HDAC5 was unchanged after PH; however HDAC5 exhibited transient nuclear accumulation in regenerating liver. These changes in hepatic HDAC expression, subcellular localization, and activity coincided with diminished histone acetylation in regenerating liver. The significance of these events was investigated by determining the effects of suberoylanilide hydroxyamic acid (SAHA, a specific inhibitor of Zn-HDAC activity) on hepatic regeneration. The results showed that SAHA-treatment suppressed the effects of PH on histone deacetylation and hepatocellular BrdU incorporation. Further examination showed that SAHA blunted hepatic expression and activation of cell cycle signals downstream of induction of cyclin D1 expression in mice subjected to PH. Conclusion The data reported here demonstrate isoform-specific regulation of Zn-HDAC expression, subcellular localization, and activity in regenerating liver. These studies also indicate that HDAC activity promotes liver regeneration by regulating hepatocellular cell cycle progression at a step downstream of cyclin D1 induction. PMID:23258575
Inhibitor of DNA binding 1 regulates cell cycle progression of endothelial progenitor cells through induction of Wnt2 expression

PubMed Central

Xia, Xi; Yu, Yang; Zhang, Li; Ma, Yang; Wang, Hong

2016-01-01

Endothelial injury is a risk factor for atherosclerosis. Endothelial progenitor cell (EPC) proliferation contributes to vascular injury repair. Overexpression of inhibitor of DNA binding 1 (Id1) significantly promotes EPC proliferation; however, the underlying molecular mechanism remains to be fully elucidated. The present study investigated the role of Id1 in cell cycle regulation of EPCs, which is closely associated with proliferation. Overexpression of Id1 increased the proportion of EPCs in the S/G2M phase and significantly increased cyclin D1 expression levels, while knockdown of Id1 arrested the cell cycle progression of EPCs in the G1 phase and inhibited cyclin D1 expression levels. In addition, it was demonstrated that Id1 upregulated wingless-type mouse mammary tumor virus integration site family member 2 (Wnt2) expression levels and promoted β-catenin accumulation and nuclear translocation. Furthermore, Wnt2 knockdown counteracted the effects of Id1 on cell cycle progression of EPCs. In conclusion, the results of the present study indicate that Id1 promoted Wnt2 expression, which accelerated cell cycle progression from G1 to S phase. This suggests that Id1 may promote cell cycle progression of EPCs, and that Wnt2 may be important in Id1 regulation of the cell cycle of EPCs. PMID:27432753
Novel Synthetic Mono-triazole Glycosides Induce G0/G1 Cell-cycle Arrest and Apoptosis in Cholangiocarcinoma Cells.

PubMed

Obchoei, Sumalee; Saeeng, Rungnapha; Wongkham, Chaisiri; Wongkham, Sopit

2016-11-01

The treatment of cholangiocarcinoma (CCA) is still ineffective and the search for a novel treatment is needed. In this study, eight novel mono-triazole glycosides (W1-W8) were synthesized and tested for their anticancer activities in CCA cell lines. The anti-proliferation effect and the underlying mechanisms of the triazole glycosides were explored. Viable cells were determined using the MTT test. Among glycosides tested, W4 and W5 exhibited the most potent anticancer activity in a dose- and time-dependent fashion. Flow cytometry and wstern blot analysis revealed that W4 and W5 induced G 0 /G 1 phase cell-cycle arrest through down-regulation of cyclin D1, cyclin E and induction of cyclin-dependent kinase inhibitors, p27 and p21 protein expression. Annexin V/propidium iodide (PI) staining demonstrated that W4 and W5 also induced apoptotic cells in a dose-dependent manner via caspase signaling cascade. Together, these findings imply that the novel synthetic glycosides might be a promising anticancer agent for CCA. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation[S

PubMed Central

Hallenborg, Philip; Petersen, Rasmus Koefoed; Feddersen, Søren; Sundekilde, Ulrik; Hansen, Jacob B.; Blagoev, Blagoy; Madsen, Lise; Kristiansen, Karsten

2014-01-01

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation. PMID:25312885
Redistribution of cell cycle by arsenic trioxide is associated with demethylation and expression changes of cell cycle related genes in acute promyelocytic leukemia cell line (NB4).

PubMed

Hassani, Saeed; Khaleghian, Ali; Ahmadian, Shahin; Alizadeh, Shaban; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2018-01-01

PML-RARα perturbs the normal epigenetic setting, which is essential to oncogenic transformation in acute promyelocytic leukemia (APL). Transcription induction and recruitment of DNA methyltransferases (DNMTs) by PML-RARα and subsequent hypermethylation are components of this perturbation. Arsenic trioxide (ATO), an important drug in APL therapy, concurrent with degradation of PML-RARα induces cell cycle change and apoptosis. How ATO causes cell cycle alteration has remained largely unexplained. Here, we investigated DNA methylation patterns of cell cycle regulatory genes promoters, the effects of ATO on the methylated genes and cell cycle distribution in an APL cell line, NB4. Analysis of promoter methylation status of 22 cell cycle related genes in NB4 revealed that CCND1, CCNE1, CCNF, CDKN1A, GADD45α, and RBL1 genes were methylated 60.7, 84.6, 58.6, 8.7, 33.4, and 73.7%, respectively, that after treatment with 2 μM ATO for 48 h, turn into 0.6, 13.8, 0.1, 6.6, 10.7, and 54.5% methylated. ATO significantly reduced the expression of DNMT1, 3A, and 3B. ATO induced the expression of CCND1, CCNE1, and GADD45α genes, suppressed the expression of CCNF and CDKN1A genes, which were consistent with decreased number of cells in G1 and S phases and increased number of cells in G2/M phase. In conclusion, demethylation and alteration in the expression level of the cell cycle related genes may be possible mechanisms in ATO-induced cell cycle arrest in APL cells. It may suggest that ATO by demethylation of CCND1 and CCNE1 and their transcriptional activation accelerates G1 and S transition into the G2/M cell cycle arrest.
Resveratrol Suppresses Growth and Migration of Myelodysplastic Cells by Inhibiting the Expression of Elevated Cyclin D1 (CCND1).

PubMed

Zhou, Wei; Xu, Shilin; Ying, Yi; Zhou, Ruiqing; Chen, Xiaowei

2017-11-01

Myelodysplastic syndromes (MDS) are a group of heterogeneous diseases characterized by poorly formed blood cells. We wanted to elucidate the underlying molecular mechanism to better determine pathogenesis, prognosis, diagnosis, and treatment for patients with MDS. We compared gene expression levels between normal and MDS tissue samples by immunohistochemical analysis. We studied the proliferation, survival, and migration of MDS cells using the EDU assay, colony formation, and transwell assays. We assessed the apoptotic rate and cell cycle status using flow cytometry and Hoechst staining. Finally, we evaluated RNA and protein expressions using polymerase chain reaction and Western blots, respectively. We found that resveratrol suppressed SKM-1 (an advanced MDS cell line) proliferation in a dose-dependent manner. Consistent with this finding, the EDU and colony formation assays also showed that resveratrol inhibited SKM-1 growth. Moreover, flow cytometry and Hoechst 33258 staining demonstrated that resveratrol induced apoptosis and a change in cell cycle status in SKM-1 cells, while the transwell assay showed that resveratrol reduced the migratory ability of SKM-1 cells. Resveratrol also decreased the expression of CCND1 (a gene that encodes the cyclin D1 protein) and increased expressions of KMT2A [lysine (K)-specific methyltransferase 2A] and caspase-3, suggesting that resveratrol exerts its effect by regulating CCND1 in SKM-1 cells. In addition, a combination of resveratrol and the PI3K/AKT inhibitor LY294002 exhibited a stronger inhibitory effect on the SKM-1 cells, compared with resveratrol alone. Our study proved that resveratrol suppresses SKM-1 growth and migration by inhibiting CCND1 expression. This finding provides novel insights into the pathogenesis of MDS and might help develop new diagnosis and treatment for patients with MDS.
TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Huang, Yong

2014-03-07

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressedmore » cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.« less
The effective application of a discrete transition model to explore cell-cycle regulation in yeast

PubMed Central

2013-01-01

Background Bench biologists often do not take part in the development of computational models for their systems, and therefore, they frequently employ them as “black-boxes”. Our aim was to construct and test a model that does not depend on the availability of quantitative data, and can be directly used without a need for intensive computational background. Results We present a discrete transition model. We used cell-cycle in budding yeast as a paradigm for a complex network, demonstrating phenomena such as sequential protein expression and activity, and cell-cycle oscillation. The structure of the network was validated by its response to computational perturbations such as mutations, and its response to mating-pheromone or nitrogen depletion. The model has a strong predicative capability, demonstrating how the activity of a specific transcription factor, Hcm1, is regulated, and what determines commitment of cells to enter and complete the cell-cycle. Conclusion The model presented herein is intuitive, yet is expressive enough to elucidate the intrinsic structure and qualitative behavior of large and complex regulatory networks. Moreover our model allowed us to examine multiple hypotheses in a simple and intuitive manner, giving rise to testable predictions. This methodology can be easily integrated as a useful approach for the study of networks, enriching experimental biology with computational insights. PMID:23915717
Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

PubMed Central

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435
The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS

PubMed Central

Duggimpudi, Sujitha; Larsson, Erik; Nabhani, Schafiq; Borkhardt, Arndt; Hoell, Jessica I

2015-01-01

Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma. PMID:25751255
p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.
Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells.

PubMed

Lachgar, S; Charveron, M; Gall, Y; Bonafe, J L

1998-03-01

The hair follicle dermal papilla which controls hair growth, is characterized in the anagen phase by a highly developed vascular network. We have demonstrated in a previous study that the expression of an angiogenic growth factor called vascular endothelial growth factor (VEGF) mRNA varied during the hair cycle. VEGF mRNA is strongly expressed in dermal papilla cells (DPC) in the anagen phase, but during the catagen and telogen phases. VEGF mRNA is less strongly expressed. This involvement of VEGF during the hair cycle allowed us to determine whether VEGF mRNA expression by DPC was regulated by minoxidil. In addition, the effect of minoxidil on VEGF protein synthesis in both cell extracts and DPC-conditioned medium, was investigated immunoenzymatically. Both VEGF mRNA and protein were significantly elevated in treated DPC compared with controls. DPC incubated with increasing minoxidil concentrations (0.2, 2, 6, 12 and 24 mumol/L) induced a dose-dependent expression of VEGF mRNA. Quantification of transcripts showed that DPC stimulated with 24 mumol/L minoxidil express six times more VEGF mRNA than controls. Similarly, VEGF protein production increases in cell extracts and conditioned media following minoxidil stimulation. These studies strongly support the likely involvement of minoxidil in the development of dermal papilla vascularization via a stimulation of VEGF expression, and support the hypothesis that minoxidil has a physiological role in maintaining a good vascularization of hair follicles in androgenetic alopecia.
Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

PubMed Central

Barna, Maria; Pusic, Aya; Zollo, Ornella; Costa, Maria; Kondrashov, Nadya; Rego, Eduardo; Rao, Pulivarthi H; Ruggero, Davide

2008-01-01

The Myc oncogene regulates the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III, and rDNA1,2. An outstanding question is whether and how increasing the cellular protein synthesis capacity can affect the multi-step process leading to cancer. We utilized ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ–Myc/+ transgenic mice to normal levels and show that in this context Myc's oncogenic potential is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a novel paradigm that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation employed to regulate the expression of selective mRNAs. We show that an aberrant increase in cap-dependent translation downstream Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (p58-PITSLRE)3-5, which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ–Myc/+ mice. When accurate translational control is re-established in Eμ–Myc/+ mice, genome instability is suppressed. Our findings reveal how perturbations in translational control provide a highly specific outcome on gene expression, genome stability, and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level. PMID:19011615
Supercritical carbon dioxide extract of Physalis peruviana induced cell cycle arrest and apoptosis in human lung cancer H661 cells.

PubMed

Wu, Shu-Jing; Chang, Shun-Pang; Lin, Doung-Liang; Wang, Shyh-Shyan; Hou, Fwu-Feuu; Ng, Lean-Teik

2009-06-01

Physalis peruviana L. (PP) is a popular folk medicine used for treating cancer, leukemia, hepatitis, rheumatism and other diseases. In this study, our objectives were to examine the total flavonoid and phenol content of different PP extracts (aqueous: HWEPP; ethanolic: EEPP; supercritical carbon dioxide: SCEPP-0, SCEPP-4 and SCEPP-5) and their antiproliferative effects in human lung cancer H661 cells. Among all the extracts tested, results showed that SCEPP-5 possessed the highest total flavonoid (226.19 +/- 4.15 mg/g) and phenol (100.82 +/- 6.25 mg/g) contents. SCEPP-5 also demonstrated the most potent inhibitory effect on H661 cell proliferation. Using DNA ladder and flow cytometry analysis, SCEPP-5 effectively induced H661 cell apoptosis as demonstrated by the accumulation of Sub-G1 peak and fragmentation of DNA. SCEPP-5 not only induced cell cycle arrest at S phase, it also up-regulated the expression of pro-apoptotic protein (Bax) and down-regulated the inhibitor of apoptosis protein (IAP). Furthermore, the apoptotic induction in H661 cells was found to associate with an elevated p53 protein expression, cytochrome c release, caspase-3 activation and PARP cleavage. Taken together, these results conclude that SCEPP-5 induced cell cycle arrest at S phase, and its apoptotic induction could be mediated through the p53-dependent pathway and modification of Bax and XIAP proteins expression. The results have also provided important pharmacological backgrounds for the potential use of PP supercritical fluid extract as products for cancer prevention.
Inhibition of mTOR's Catalytic Site by PKI-587 Is a Promising Therapeutic Option for Gastroenteropancreatic Neuroendocrine Tumor Disease

PubMed Central

Freitag, Helma; Christen, Friederike; Lewens, Florentine; Grass, Irina; Briest, Franziska; Iwaszkiewicz, Sara; Siegmund, Britta; Grabowski, Patricia

2017-01-01

Background The characteristic clinical heterogeneity and mostly slow-growing behavior of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) cause problems in finding appropriate treatments. Thus, the current therapy options are not satisfactory. PKI-587 is a highly potent, novel dual inhibitor of PI3K and mTORC1/C2. Aim We assessed the effects of PKI-587 in different GEP-NEN tumor models, including the poorly differentiated cell line LCC-18, and compared them with those of the established mTORC1 inhibitor everolimus. Methods We treated BON, QGP-1, KRJ-I, and LCC-18 cell lines with increasing concentrations of the inhibitor PKI-587, and compared the results with those of everolimus and DMSO. We assessed the impact of the treatments on viability (WST-1 assay), on apoptotic processes (caspase 3/7 assay, JC-1), and on cell cycle regulation (flow cytometry). We determined alterations in signaling mediators by phosphor-specific Western blot analysis and conducted multiplexed gene expression analysis (nCounter® technology). Results In all cell lines, PKI-587 dose-dependently inhibited proliferation, whereas everolimus was less effective. Treatment with PKI-587 led to cell cycle arrest and induction of apoptosis and successfully suppressed activity of the direct mTORC1 target 4E-BP1, a crucial factor for tumor genesis only partially inhibited by everolimus. Gene expression analyses revealed relevant changes of RAS, MAPK, STAT, and PI3K pathway genes after treatment. Treatment-dependent and cell line-characteristic effects on AKT/Rb/E2F signaling regarding cell cycle control and apoptosis are extensively discussed in this paper. Conclusion PI3K/mTOR dual targeting is a promising new therapeutic approach in neuroendocrine tumor disease that should be evaluated in further clinical trials. PMID:27513674
Dose-Dependent Dual Role of PIT-1 (POU1F1) in Somatolactotroph Cell Proliferation and Apoptosis

PubMed Central

Jullien, Nicolas; Roche, Catherine; Brue, Thierry; Figarella-Branger, Dominique; Graillon, Thomas; Barlier, Anne; Herman, Jean-Paul

2015-01-01

To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called “dominant-negative” action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1. PMID:25822178

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study.

PubMed

Skowronska, Agnieszka; Młotkowska, Patrycja; Wojciechowicz, Bartosz; Okrasa, Stanisław; Nielsen, Soren; Skowronski, Mariusz T

2015-02-18

The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. Uterine tissues were collected on Days 10-12 and 14-16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.
Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less
The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Bögler, Oliver; Duchrow, Michael

2003-01-01

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase. Copyright 2002 John Wiley & Sons, Ltd.
Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH.

PubMed

Grazul-Bilska, Anna T; Thammasiri, Jiratti; Kraisoon, Aree; Reyaz, Arshi; Bass, Casie S; Kaminski, Samantha L; Navanukraw, Chainarong; Redmer, Dale A

2018-03-01

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions. Copyright © 2017 Elsevier Inc. All rights reserved.
Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis.

PubMed

Kim, Hyoji; Choi, Hoyun; Lee, Suk Kyeong

2016-02-01

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Bone Morphogenetic Protein Regulation of Enteric Neuronal Phenotypic Diversity: Relationship to Timing of Cell Cycle Exit

PubMed Central

Chalazonitis, Alcmène; Pham, Tuan.D.; Li, Zhishan; Roman, Daniel; Guha, Udayan; Gomes, William; Kan, Lixin; Kessler, John A.; Gershon, Michael D.

2008-01-01

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice over expressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (γ-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene related peptide, TrkC). Numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development. PMID:18537141
Cell cycle re-entry sensitizes podocytes to injury induced death.

PubMed

Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

2016-07-17

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.
Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

PubMed

Naselli, Flores; Tesoriere, Luisa; Caradonna, Fabio; Bellavia, Daniele; Attanzio, Alessandro; Gentile, Carla; Livrea, Maria A

2014-07-18

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells. Copyright © 2014 Elsevier Inc. All rights reserved.
Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

PubMed

Cen, Yan-Hui; Guo, Wen-Wen; Luo, Bin; Lin, Yong-Da; Zhang, Qing-Mei; Zhou, Su-Fang; Luo, Guo-Rong; Xiao, Shao-Wen; Xie, Xiao-Xun

2012-10-01

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.
An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA

PubMed Central

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O.; Linne, Uwe; Albaum, Stefan P.; Jiménez-Zurdo, José I.; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans-encoded sRNA (trans-sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression. PMID:29740411
An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

PubMed

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression.
Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

PubMed

Mavri-Damelin, Demetra; Eaton, Simon; Damelin, Leonard H; Rees, Myrddin; Hodgson, Humphrey J F; Selden, Clare

2007-01-01

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.
Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Wei-Hong; Yang, Li-Yun; Cao, Zhong-Yi, E-mail: m18070383032@163.com

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. Inmore » all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC. - Highlights: • Tivantinib suppresses OSCC cell growth independent of the inhibition of HGF/MET signaling pathway. • Tivantinib blocks cell cycle and induces caspases-mediated apoptosis. • Tivantinib elicits its anti-tumor activity with the inhibition of FAK signaling pathway.« less
Alpinia oxyphylla Miq. fruit extract activates IGFR-PI3K/Akt signaling to induce Schwann cell proliferation and sciatic nerve regeneration.

PubMed

Chang, Yung-Ming; Chang, Hen-Hong; Tsai, Chin-Chuan; Lin, Hung-Jen; Ho, Tsung-Jung; Ye, Chi-Xin; Chiu, Ping-Ling; Chen, Yueh-Sheng; Chen, Ray-Jade; Huang, Chih-Yang; Lin, Chien-Chung

2017-03-31

It is known that the medicinal herb Alpinia oxyphylla Miq. is widely used as a remedy for diarrhea as well as the symptoms accompanying hypertension and cerebrovascular disorders. Moreover, it has also been reported that Alpinia oxyphylla Miq. has beneficial effects on anti-senescence and neuro-protection. This study focuses on the molecular mechanisms by which the Alpinia oxyphylla Miq. fruits promote neuron regeneration. A piece of silicone rubber was guided across a 15 mm gap in the sciatic nerve of a rat. This nerve gap was then filled with various doses of Alpinia oxyphylla Miq. fruits to assess their regenerative effect on damaged nerves. Further, we investigated the role of Alpinia oxyphylla Miq. fruits in RSC96 Schwann cell proliferation. Our current results showed that treatment with the extract of Alpinia oxyphylla Miq. fruits triggers the phosphorylated insulin-like growth factor-1 receptor- phosphatidylinositol 3-kinase/serine-threonine kinase pathway, and up-regulated the proliferating cell nuclear antigen in a dose-dependent manner. Cell cycle analysis on RSC96 Schwann cells showed that, after exposure to Alpinia oxyphylla Miq. fruit extract, the transition from the first gap phase to the synthesis phase occurs in 12-18 h. The expression of the cell cycle regulatory proteins cyclin D1, cyclin E and cyclin A increased in a dose-dependent manner. Transfection with a small interfering RNA blocked the expression of phosphatidylinositol 3-kinase and induced down-regulation both on the mRNA and protein levels, which resulted in a reduction of the expression of the survival factor B-cell lymphoma 2. We provide positive results that demonstrate that Alpinia oxyphylla Miq. fruits facilitate the survival and proliferation of RSC96 cells via insulin-like growth factor-1 signaling.
A stem cell apostasy: A tale of 4 H words

PubMed Central

Quesenberry, Peter J.; Goldberg, Laura R.; Dooner, Mark S.

2014-01-01

The field of hematopoietic stem cell biology has become increasingly dominated by the pursuit and study of highly purified populations of hematopoietic stem cells (HSCs). Such HSCs are typically isolated based on their cell surface marker expression patterns and ultimately defined by their multipotency and capacity for self-generation. However, even with progressively more stringent stem cell separation techniques, the resultant HSC population remains heterogeneous with respect to both self-renewal and differentiation capacity. Critical studies on un-separated whole bone marrow (WBM) have definitively shown that long-term engraftable hematopoietic stem cells are in active cell cycle and thus continually changing phenotype. Therefore, they cannot be purified by current approaches dependent on stable surface epitope expression because the surface markers are continually changing as well. These critical cycling cells are discarded with current stem cell purifications. Despite this, research defining such characteristics as self-renewal capacity, lineage-commitment, bone marrow niches, and proliferative state of HSCs continues to focus predominantly on this small sub-population of purified marrow cells. This review discusses the research leading to the hierarchical model of hematopoiesis and questions the dogmas pertaining to HSC quiescence and purification. PMID:25183450
Impact of CD40 expression by flowcytometry on outcome of patients with non-Hodgkin's lymphoma.

PubMed

Soliman, Mohamed A; Fathy, Amr Ahmed; Alkilani, Amira; Abd El-Bary, Naser; El-Bassal, Fathai

2009-01-01

Lymphoid malignancies represent a wide variety of disease entities characterized by malignant proliferation of lymphoid cells which have distinct clinical features, cellular morphology, immunophenotype, cytogenetic changes and histologic features. CD40 is a member of the tumor necrosis factor receptor super-family. It was first identified and characterized in B cell, signaling through the CD40 receptor was found to play an important role in multiple events in T-cell dependent antibody response including B-cell survival and proliferation, memory B-cell formation and immunoglobulin isotype switching. The aim of this study is to detect the expression of CD40 on B lymphocytes in patients suffering from Non-Hodgkin's Lymphoma and correlate the results with the patients' response to treatment protocols. This study was carried out on 114 patients, of them only 100 patients completed 4 cycles of chemotherapy and were valuable. Their age was ranged from 17 to 63 years old. Fifteen age and gender matched individuals were, also, selected as a control group. CD40 expression was measured on peripheral blood samples by flowcytometry at patient's presentation as well as after 4 cycles of chemotherapy. This study showed that there's significant decrease in the mean values of % of CD40 on B-cell in patients with NHL in all stages when compared with normal control group. Also the study showed that there's statistical significant correlation between percent of CD40 on B-lymphocytes and stage of lymphoma, i.e., the more advanced stage, the lower the % of CD40 on B-cell. After receiving a corresponding treatment, the CD40 expression is increased in significant correlation with the response to treatment. (This is a preliminary result after 4 cycles of CHOP treatment). We concluded that CD40 Lymphocyte development occurs in discrete functional steps that are defined by the onset of expression is highly expressed in healthy subjects and its expression on B-lymphocyte is decreased with advanced stage of NHL. Percent of CD40 on B-lymphocyte can be considered as an evaluation marker for outcome of treatment in NHL patients as its expression is increased in responding patients.
Ruthenium complex Λ-WH0402 induces hepatocellular carcinoma LM6 (HCCLM6) cell death by triggering the Beclin-1-dependent autophagy pathway.

PubMed

Yuan, Jian; Lei, Zhinian; Wang, Xi; Zhu, Feng; Chen, Dongbo

2015-05-01

To evaluate the anticancer mechanism of the new ruthenium complex-Λ-WH0402 at the cellular level, the in vitro cytotoxicity of Λ-WH0402 was investigated on 10 human tumor cell lines. Λ-WH0402 was found to have higher anticancer activity than cisplatin toward human liver cancer HCCLM6 cells that have high tumor metastatic characteristics. Meanwhile, Λ-WH0402 showed an antimetastatic effect on HCCLM6 cells in vitro, mostly through its effect on cell adhesion, invasion and migration. In addition, Λ-WH0402 significantly reduced tumor metastasis to the lungs in orthotopic mouse hepatocellular cancer (HCC) models induced by HCCLM6 cells. Furthermore, Λ-WH0402 exerted an inhibitory effect on tumor cell growth and proliferation and induced dose-dependent cell cycle arrest in the S phase in HCCLM6 cells. Immunoblotting analysis showed that Λ-WH0402 not only decreased the expression of antiapoptotic protein Bcl-2 and nutrient-deprivation autophagy factor-1 (NAF-1), but also significantly increased the expression of Beclin-1 in HCCLM6 cells. More importantly, we identified that Λ-WH0402 treatment reduced the interaction between Bcl-2 and Beclin-1, and increased the expression of autophagic activation marker LC3B-II in HCCLM6 cells. On the whole, our results suggested that the anitcancer activity of Λ-WH0402 is mediated through promoting the Beclin-1-dependent autophagy pathway in HCCLM6 cells.
Cell Cycle-Dependent Phosphorylation of Theileria annulata Schizont Surface Proteins

PubMed Central

von Schubert, Conrad; Wastling, Jonathan M.; Heussler, Volker T.; Woods, Kerry L.

2014-01-01

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state. PMID:25077614
The renal cell carcinoma-associated oncogenic fusion protein PRCCTFE3 provokes p21{sup W{sup A{sup F{sup 1{sup /{sup C{sup I{sup P{sup 1}}}}}}}}}-mediated cell cycle delay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medendorp, Klaas; Groningen, Jan J.M. van; Vreede, Lilian

2009-08-15

Previously, we found that in t(X;1)(p11;q21)-positive renal cell carcinomas the bHLH-LZ transcription factor TFE3 is fused to a novel protein designated PRCC. In addition, we found that the PRCCTFE3 fusion protein, which has retained all known functional domains of TFE3, acts as a more potent transcriptional activator than wild type TFE3. We also found that PRCCTFE3 expression confers in vitro and in vivo transformation onto various cell types, including those of the kidney. Here we show that de novo expression of the PRCCTFE3 fusion protein provokes cell cycle delay. This delay, which is mediated by induction of the cyclin-dependent kinasemore » inhibitor p21{sup W{sup A{sup F{sup 1{sup /{sup C{sup I{sup P{sup 1}}}}}}}}}, affects both the G1/S and the G2/M phases of the cell cycle and prevents the cells from undergoing polyploidization. We also show that the PRCCTFE3 fusion protein binds directly to the p21{sup W{sup A{sup F{sup 1{sup /{sup C{sup I{sup P{sup 1}}}}}}}}} promoter and that the PRCCTFE3-induced up-regulation of p21{sup W{sup A{sup F{sup 1{sup /{sup C{sup I{sup P{sup 1}}}}}}}}} leads to activation of the pRB pathway. Finally, we show that in t(X;1)(p11;q21)-positive renal tumor cells several processes that link PRCCTFE3 expression to p21{sup W{sup A{sup F{sup 1{sup /{sup C{sup I{sup P{sup 1}}}}}}}}}-mediated cell cycle delay are abrogated. Our data suggest a scenario in which, during the course of renal cell carcinoma development, an initial PRCCTFE3-induced cell cycle delay must be numbed, thus permitting continued proliferation and progression towards full-blown malignancy.« less
Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.
The roles of p53R2 in cancer progression based on the new function of mutant p53 and cytoplasmic p21.

PubMed

Yousefi, Bahman; Rahmati, Mohammad; Ahmadi, Yasin

2014-03-18

Although the deregulated expression of p53R2, a p53-inducible protein and homologue of the R2 subunit of ribonucleotide reductase, has been detected in several human cancers, p53R2 roles in cancer progression and malignancy still remains controversial. In this article, we present a viable hypothesis about the roles of p53R2 in cancer progression and therapy resistance based on the roles of cytoplasmic p21 and mutant p53. Since p53R2 can up-regulate p21 and p21, it in turn has a dual role in cell cycle. Hence, p53R2 can play a dual role in cell cycle progression. In addition, because p53 is the main regulator of p53R2, the mutant p53 may induce the expression of p53R2 in some cancer cells based on the "keep of function" phenomenon. Therefore, depending on the locations of p21 and the new abilities of mutant p53, p53R2 has dual role in cell cycle progression. Since the DNA damaging therapies induce p53R2 expression through the induction of p53, p53R2 can be the main therapy resistance mediator in cancers with cytoplasmic p21. Copyright © 2014 Elsevier Inc. All rights reserved.
5-epi-Sinuleptolide induces cell cycle arrest and apoptosis through tumor necrosis factor/mitochondria-mediated caspase signaling pathway in human skin cancer cells.

PubMed

Liang, Chia-Hua; Wang, Guey-Horng; Chou, Tzung-Han; Wang, Shih-Hao; Lin, Rong-Jyh; Chan, Leong-Perng; So, Edmund Cheung; Sheu, Jyh-Horng

2012-07-01

Skin cancers are reportedly increasing worldwide. Developing novel anti-skin cancer drugs with minimal side effects is necessary to address this public health issue. Sinuleptolide has been demonstrated to possess anti-cancer cell activities; however, the mechanisms underlying the anti-skin cancer effects of 5-epi-sinuleptolide and sinuleptolide remain poorly understood. Apoptosis cell, cell-cycle-related regulatory factors, and mitochondria- and death receptor-dependent caspase pathway in 5-epi-sinuleptolide-induced cell apoptosis were examined using SCC25 cells. 5-epi-Sinuleptolide inhibited human skin cancer cell growth more than did sinuleptolide. Treatment of SCC25 cells with 5-epi-sinuleptolide increased apoptotic body formation, and induced cell-cycle arrest during the G2/M phase. Notably, 5-epi-sinuleptolide up-regulated p53 and p21 expression and inhibited G2/M phase regulators of cyclin B1 and cyclin-dependent kinease 1 (CDK1) in SCC25 cells. Additionally, 5-epi-sinuleptolide induced apoptosis by mitochondria-mediated cytochrome c and Bax up-expression, down-regulated Bcl-2, and activated caspase-9 and -3. 5-epi-Sinuleptolide also up-regulated tBid, which is associated with up-regulation of tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) and their cognate receptors (i.e., TNF-RI, TNF-R2 and Fas), downstream adaptor TNF-R1-associated death domain (TRADD) and Fas-associated death domain (FADD), and activated caspase-8 in SCC25 cells. The analytical results indicate that the death receptor- and mitochondria-mediated caspase pathway is critical in 5-epi-sinuleptolide-induced apoptosis of skin cancer cells. This is the first report suggesting that the apoptosis mediates the anti-tumor effect of 5-epi-sinuleptolide. The results of this study might provide useful suggestions for designing of anti-tumor drugs for skin cancer patients. Copyright © 2012 Elsevier B.V. All rights reserved.
Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

PubMed

Lin, Shuw-Yuan; Lai, Wan-Wen; Chou, Chi-Chung; Kuo, Hsiu-Maan; Li, Te-Mao; Chung, Jing-Gung; Yang, Jen-Hung

2006-12-01

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.
Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

PubMed

Zhu, Qiaohua; Yu, Xinfa; Zhou, Zhi-Wei; Zhou, Chengyu; Chen, Xiao-Wu; Zhou, Shu-Feng

2017-01-01

Aurora A kinase represent a feasible target in cancer therapy. To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Analysis of uncultured extremophilic snow algae by non-invasive single cell Raman spectroscopy

NASA Astrophysics Data System (ADS)

Beer, Thomas; Tanaka, Zuki; Netzter, Nathan; Rothschild, Lynn J.; Chen, Bin

2011-10-01

The study of life in extreme environments is a critical component of Astrobiology. But many of the so-called "extremophiles" are not readily cultivatable and therefore difficult to study under laboratory conditions. An example of such an extremophile is the snow alga Chlamydomonas cd. nivalis which expresses still unstudied secondary metabolites within its life cycle. In this paper, we present the first time the non-invasive single cell Raman spectroscopy of the life cycle dependent metabolite composition of C. nivalis. These secondary metabolites are likely related to the adaptation of C. nivalis to various stress factors. Normalized carotenoid Raman spectra intensities reveal characteristic ratio differences that allow identification of life cycle stages and putative secondary metabolites.
CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

PubMed

He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

2016-01-01

The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.
Study on the Mechanism of Cell Cycle Checkpoint Kinase 2 (CHEK2) Gene Dysfunction in Chemotherapeutic Drug Resistance of Triple Negative Breast Cancer Cells.

PubMed

Luo, Li; Gao, Wei; Wang, Jinghui; Wang, Dingxue; Peng, Xiaobo; Jia, Zhaoyang; Jiang, Ye; Li, Gongzhuo; Tang, Dongxin; Wang, Yajie

2018-05-15

BACKGROUND This study aimed to investigate the mechanism of CHEK2 gene dysfunction in drug resistance of triple negative breast cancer (TNBC) cells. MATERIAL AND METHODS To perform our study, a stable CHEK2 wild type (CHEK2 WT) or CHEK2 Y390C mutation (CHEK2 Y390C) expressed MDA-MB-231 cell line was established. MTT assay, cell apoptosis assay and cell cycle assay were carried out to analyze the cell viability, apoptosis, and cell cycle respectively. Western blotting and qRT-PCR were applied for related protein and gene expression detection. RESULTS We found that the IC50 value of DDP (Cisplatin) to CHEK2 Y390C expressed MDA-MB-231 cells was significantly higher than that of the CHEK2 WT expressed cells and the control cells. After treatment with DDP for 48 h, cells expressing CHEK2 WT showed lower cell viability than that of the CHEK2 Y390C expressed cells and the control cells; compared with the CHEK2 Y390C expressed cells and the control cells, cells expressing CHEK2 WT showed significant G1/S arrest. Meanwhile, we found that compared with the CHEK2 Y390C expressed cells and the control cells, cell apoptosis was significantly increased in CHEK2 WT expressed cells. Moreover, our results suggested that cells expressing CHEK2 WT showed higher level of p-CDC25A, p-p53, p21, Bax, PUMA, and Noxa than that of the CHEK2 Y390C expressed cells and the control cells. CONCLUSIONS Our findings indicated that CHEK2 Y390C mutation induced the drug resistance of TNBC cells to chemotherapeutic drugs through administrating cell apoptosis and cell cycle arrest via regulating p53 activation and CHEK2-p53 apoptosis pathway.
Curcumin induces apoptosis in pancreatic cancer cells through the induction of forkhead box O1 and inhibition of the PI3K/Akt pathway.

PubMed

Zhao, Zhiming; Li, Chenggang; Xi, Hao; Gao, Yuanxing; Xu, Dabin

2015-10-01

Previous population investigations have suggested that the application of curcumin may be associated with decreased incidence and improved prognosis in certain types of cancer. Forkhead box O1 (FOXO1) has been implicated in the regulation of several biological processes, including stress resistance, metabolism, DNA repair, cell cycle and apoptosis. The aims of the present study were to investigate the effects and molecular mechanisms of curcumin on the induction of anti‑proliferation, cell cycle arrest and apoptosis, by FOXO1, in pancreatic cancer cells. The MTT assay and ELISA‑Brdu assay were used to assess cell proliferation. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect the expression of PCNA, Ki‑67, B‑cell lymphoma‑2 (Bcl‑2), B‑cell‑associated X protein (Bax), cyclin D1, p21, p27 and FOXO1. Cell apoptosis was detected using a Cell Death ELISA detection kit. A Caspase‑3/9 Fluorescent Assay kit was used to detect caspase activity. The findings revealed that curcumin significantly decreased cell proliferation, which was associated with increased expression of the p21/CIP1 and p27/KIP1 cyclin‑dependent kinase inhibitors, and inhibited expression of cyclin D1. In addition, curcumin induced apoptosis by decreasing the Bcl‑2/Bax protein ratio and increasing caspase‑9/3 activation in the pancreatic cancer cells. Using siRNA against FOXO1, and Akt inhibitor and activator, the present study confirmed that curcumin induced the expression of FOXO1 by inhibition of phosphoinositide 3‑kinase/Akt signaling, leading to cell cycle arrest and apoptosis. In conclusion, these findings offer support for a mechanism that may underlie the anti‑neoplastic effects of curcumin and justify further investigation to examine the potential roles for activators of FOXO1 in the prevention and treatment of pancreatic cancer.
12-Chloracetyl-PPD, a novel dammarane derivative, shows anti-cancer activity via delay the progression of cell cycle G2/M phase and reactive oxygen species-mediate cell apoptosis.

PubMed

Wang, Xu De; Sun, Yuan Yuan; Zhao, Chen; Qu, Fan Zhi; Zhao, Yu Qing

2017-03-05

(20R)-Dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) is a ginsenoside isolated from Panax ginseng (C. A. Meyer). This compound exhibits anti-cancer activities on many human cancer cell lines. In this study, we investigated anti-cancer mechanisms of 12β-O-( L -Chloracetyl)-dammar-20(22)-ene-3β,25-diol(12-Chloracetyl-PPD), a modified 25-OH-PPD. We found that compound 12-Chloracetyl-PPD resulted in a concentration-dependent inhibition of viability in prostate, breast, and gastric cancer cells, without affecting the viability of normal cell (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2). In MDA-MB-435 and C4-2B cancer cells, 12-Chloracetyl-PPD induced G2/M cell cycle arrest, down-regulated mouse double minute 2 (MDM2) expression, up-regulated p53 expression, triggered apoptosis, and stimulated reactive oxygen species production. Apoptosis can be attenuated by the reactive oxygen species scavenger N-acetylcysteine. Our results suggested that compound 12-Chloracetyl-PPD showed obvious anti-cancer activity based on delaying cell cycle arrest and inducing cell apoptosis by reactive oxygen species production, which supported development of 12-Chloracetyl-PPD as a potential agent for cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.
Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

PubMed

Haoudi, Abdelali; Daniels, Rodney C; Wong, Eric; Kupfer, Gary; Semmes, O John

2003-09-26

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.
Expression and function of activin receptors in human endometrial adenocarcinoma cells.

PubMed

Tanaka, Tetsuji; Toujima, Saori; Otani, Tsutomu; Minami, Sawako; Yamoto, Mareo; Umesaki, Naohiko

2003-09-01

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.
G protein-coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis.

PubMed

Mori, Taisuke; Ito, Fumitake; Matsushima, Hiroshi; Takaoka, Osamu; Tanaka, Yukiko; Koshiba, Akemi; Kusuki, Izumi; Kitawaki, Jo

2015-05-01

To demonstrate the effects of the selective G protein-coupled estrogen receptor 1 (GPER) agonist G-1 in human ovarian endometriotic stromal cells (ESCs). Experimental in vitro study. University hospital. A total of 33 patients with ovarian endometrioma. Endometriotic stromal cells from ovarian chocolate cysts were treated with the GPER agonist G-1. The primary outcomes were cell proliferation, measured using the WST-8 assay; cell cycle, as analyzed using flow cytometry, fluorescent immunocytochemistry, and cytotoxicity; caspase activity, as measured by fluorescent and luminescent enzyme assays; and protein expression levels, as determined by Western blot analysis. G-1 suppressed ESC proliferation in a concentration-dependent manner. The inhibitory effect was not blocked when GPER signaling pathways, including the GPER itself, were inhibited. G-1 induced cell cycle arrest and accumulation in the sub-G1 phase in ESCs. Immunofluorescence analysis demonstrated that G-1 interrupted microtubule assembly at the mitotic phase. G-1 also induced caspase-3-dependent apoptosis without significant cytotoxicity. G-1 suppressed proliferation and induced apoptosis in ESCs, suggesting the potential use of this compound as a therapeutic drug for the treatment of endometriosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition

PubMed Central

Fu, Changlong; Zheng, Chunsong; Lin, Jie; Ye, Jinxia; Mei, Yangyang; Pan, Caibin; Wu, Guangwen; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2017-01-01

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose- and time-dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin-dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis. PMID:28358416
13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.

PubMed

Nelson, Amanda M; Gilliland, Kathryn L; Cong, Zhaoyuan; Thiboutot, Diane M

2006-10-01

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.
DEVELOPMENTAL NEUROTOXICITY OF ORGANOPHOSPHATES TARGETS CELL CYCLE AND APOPTOSIS, REVEALED BY TRANSCRIPTIONAL PROFILES IN VIVO AND IN VITRO

PubMed Central

Slotkin, Theodore A.; Seidler, Frederic J.

2012-01-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1–4, in doses straddling the threshold for barely-detectable cholinesterase, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20–25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60–70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. PMID:22222554
Developmental neurotoxicity of organophosphates targets cell cycle and apoptosis, revealed by transcriptional profiles in vivo and in vitro.

PubMed

Slotkin, Theodore A; Seidler, Frederic J

2012-03-01

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1-4, in doses straddling the threshold for barely-detectable cholinesterase inhibition, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20-25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60-70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme. Copyright Â© 2011 Elsevier Inc. All rights reserved.
NeoPalAna: Neoadjuvant palbociclib, a cyclin-dependent kinase 4/6 inhibitor, and anastrozole for clinical stage 2 or 3 estrogen receptor positive breast cancer

PubMed Central

Ma, Cynthia X.; Gao, Feng; Luo, Jingqin; Northfelt, Donald W.; Goetz, Matthew; Forero, Andres; Hoog, Jeremy; Naughton, Michael; Ademuyiwa, Foluso; Suresh, Rama; Anderson, Karen S.; Margenthaler, Julie; Aft, Rebecca; Hobday, Timothy; Moynihan, Timothy; Gillanders, William; Cyr, Amy; Eberlein, Timothy J.; Hieken, Tina; Krontiras, Helen; Guo, Zhanfang; Lee, Michelle V.; Spies, Nicholas C.; Skidmore, Zachary L.; Griffith, Obi L.; Griffith, Malachi; Thomas, Shana; Bumb, Caroline; Vij, Kiran; Bartlett, Cynthia Huang; Koehler, Maria; Al-Kateb, Hussam; Sanati, Souzan; Ellis, Matthew J.

2017-01-01

Purpose Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the anti-proliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies. Experimental Design Eligible patients with clinical stage II/III ER+/HER2- breast cancer received anastrozole 1mg daily for 4 weeks (cycle 0) (with goserelin if premenopausal), followed by adding palbociclib (125mg daily on days 1-21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67>10%, in which case patients went off study due to inadequately response. Anastrozole was continued until surgery, which occurred 3-5 weeks post palbociclib exposure. Later patients received additional 10-12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression and mutation profiles. The primary endpoint was Complete Cell Cycle Arrest (CCCA: central Ki67<2.7%). Results Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs C1D1 26%, p<0.001). Palbociclib enhanced cell cycle control over anastrozole monotherapy regardless of luminal subtype (A vs B) and PIK3CA status with activity observed across a broad range of clinicopathological and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with non-luminal subtypes and persistent E2F-target gene expression. Conclusions Palbociclib is an active anti-proliferative agent for early-stage breast cancer resistant to anastrozole, however, prolonged administration may be necessary to maintain its effect. PMID:28270497
Targeting c-Myc: JQ1 as a promising option for c-Myc-amplified esophageal squamous cell carcinoma.

PubMed

Wang, Jingyuan; Liu, Zhentao; Wang, Ziqi; Wang, Shubin; Chen, Zuhua; Li, Zhongwu; Zhang, Mengqi; Zou, Jianling; Dong, Bin; Gao, Jing; Shen, Lin

2018-04-10

c-Myc amplification-induced cell cycle dysregulation is a common cause for esophageal squamous cell carcinoma (ESCC), but no approved targeted drug is available so far. The bromodomain inhibitor JQ1, which targets c-Myc, exerts anti-tumor activity in multiple cancers. However, the role of JQ1 in ESCC remains unknown. In this study, we reported that JQ1 had potent anti-proliferative effects on ESCC cells in both time- and dose-dependent manners by inducing cell cycle arrest at G1 phase, cell apoptosis, and the mesenchymal-epithelial transition. Follow-up studies revealed that both c-Myc/cyclin/Rb and PI3K/AKT signaling pathways were inactivated by JQ1, as indicated by the downregulation of c-Myc, cyclin A/E, and phosphorylated Rb, AKT and S6. Tumor suppression induced by JQ1 in c-Myc amplified or highly expressed xenografts was higher than that in xenografts with low expression, suggesting its potential role in prediction. In conclusion, targeting c-Myc by JQ1 could cause significant tumor suppression in ESCC both in vitro and in vivo. Also, c-Myc amplification or high expression might serve as a potential biomarker and provide a promising therapeutic option for ESCC. Copyright © 2018 Elsevier B.V. All rights reserved.
A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hwa-Ryeon; Roe, Jae-Seok; Lee, Ji-Eun

2012-02-24

Highlights: Black-Right-Pointing-Pointer p53 downregulates IMPDH. Black-Right-Pointing-Pointer p53-dependent miR-34a transactivation inhibits IMPDH transcription. Black-Right-Pointing-Pointer miR-34a-mediated inhibition of IMPDH downregulates GTP-dependent Ras signal. -- Abstract: p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5 Primemore » -monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.« less

Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway

PubMed Central

Yuan, Lingqin; Sheng, Xiugui; Willson, Adam K; Roque, Dario R; Stine, Jessica E; Guo, Hui; Jones, Hannah M; Zhou, Chunxiao; Bae-Jump, Victoria L

2015-01-01

Glutamine is one of the main nutrients used by tumor cells for biosynthesis. Therefore, targeted inhibition of glutamine metabolism may have anti-tumorigenic implications. In the present study, we aimed to evaluate the effects of glutamine on ovarian cancer cell growth. Three ovarian cancer cell lines, HEY, SKOV3, and IGROV-1, were assayed for glutamine dependence by analyzing cytotoxicity, cell cycle progression, apoptosis, cell stress, and glucose/glutamine metabolism. Our results revealed that administration of glutamine increased cell proliferation in all three ovarian cancer cell lines in a dose dependent manner. Depletion of glutamine induced reactive oxygen species and expression of endoplasmic reticulum stress proteins. In addition, glutamine increased the activity of glutaminase (GLS) and glutamate dehydrogenase (GDH) by modulating the mTOR/S6 and MAPK pathways. Inhibition of mTOR activity by rapamycin or blocking S6 expression by siRNA inhibited GDH and GLS activity, leading to a decrease in glutamine-induced cell proliferation. These studies suggest that targeting glutamine metabolism may be a promising therapeutic strategy in the treatment of ovarian cancer. PMID:26045471
Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.
Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.
Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschläger, Markus

2006-07-15

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.
FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

PubMed

Sewastianik, T; Szydlowski, M; Jablonska, E; Bialopiotrowicz, E; Kiliszek, P; Gorniak, P; Polak, A; Prochorec-Sobieszek, M; Szumera-Cieckiewicz, A; Kaminski, T S; Markowicz, S; Nowak, E; Grygorowicz, M A; Warzocha, K; Juszczynski, P

2016-11-17

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.
Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression

PubMed Central

Ye, Jian; Yao, Yufeng; Song, Qixue; Li, Sisi; Hu, Zhenkun; Yu, Yubing; Hu, Changqing; Da, Xingwen; Li, Hui; Chen, Qiuyun; Wang, Qing K.

2016-01-01

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. To elucidate new regulatory mechanisms for heptocarcinogenesis, we investigated the regulation of p21, a cyclin-dependent kinase (CDK) inhibitor encoded by CDKN1A, in HCC. The expression level of p21 is decreased with the progression of HCC. Luciferase assays with a luciferase-p21-3′ UTR reporter and its serial deletions identified a 15-bp repressor element at the 3′-UTR of CDKN1A, which contains a binding site for miR-95-3p. Mutation of the binding site eliminated the regulatory effect of miR-95-3p on p21 expression. Posttranscriptional regulation of p21 expression by miR-95-3p is mainly on the protein level (suppression of translation). Overexpression of miR-95-3p in two different HCC cell lines, HepG2 and SMMC7721, significantly promoted cell proliferation, cell cycle progression and cell migration, whereas a miR-95-3p specific inhibitor decreased cell proliferation, cell cycle progression and cell migration. The effects of miR-95-3p on cellular functions were rescued by overexpression of p21. Overexpression of miR-95-3p promoted cell proliferation and tumor growth in HCC xenograft mouse models. Expression of miR-95-3p was significantly higher in HCC samples than in adjacent non-cancerous samples. These results demonstrate that miR-95-3p is a potential new marker for HCC and regulates hepatocarcinogenesis by directly targeting CDKN1A/p21 expression. PMID:27698442
p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijjar, Tarlochan; Wigington, Don; Garbe, James C.

1999-08-01

The authors have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMEC). HMEC immortalized following chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined and exhibited slow heterogeneous growth, and contained many non-proliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very graduall2048nverted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerasemore » activity, and stabilization of telomere length. The fully immortal good growing HMEC did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele, and transient expression of the previously imprinted allele. Conditionally immortal 184A1 with mean TRF > 3 kb infected with retroviruses containing the p57 gene exhibited premature slow heterogeneous growth. Conversely, exogenous expression of hTERT, the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMEC which have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57 mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)

Gabrielson, Marike; Reizer, Edwin; Stål, Olle

An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclearmore » Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G{sub 1}-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G{sub 1}-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. - Highlights: • Proposed cell cycle regulation through the mitochondrial transporter SLC25A43. • SLC25A43 alters cell proliferation rate and cell cycle progression. • Suppressed SLC25A43 influences transcription of cell cycle regulatory genes.« less
Sulforaphane inhibits PDGF-induced proliferation of rat aortic vascular smooth muscle cell by up-regulation of p53 leading to G1/S cell cycle arrest.

PubMed

Yoo, Su-Hyang; Lim, Yong; Kim, Seung-Jung; Yoo, Kyu-Dong; Yoo, Hwan-Soo; Hong, Jin-Tae; Lee, Mi-Yea; Yun, Yeo-Pyo

2013-01-01

Vascular diseases such as atherosclerosis and restenosis artery angioplasty are associated with vascular smooth muscle cell (VSMC) proliferation and intimal thickening arterial walls. In the present study, we investigated the inhibitory effects of sulforaphane, an isothiocyanate produced in cruciferous vegetables, on VSMC proliferation and neointimal formation in a rat carotid artery injury model. Sulforaphane at the concentrations of 0.5, 1.0, and 2.0 μM significantly inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation in a concentration-dependent manner, determined by cell count. The IC50 value of sulforaphane-inhibited VSMC proliferation was 0.8 μM. Sulforaphane increased the cyclin-dependent kinase inhibitor p21 and p53 levels, while it decreased CDK2 and cyclin E expression. The effects of sulforaphane on vascular thickening were determined 14 days after the injury to the rat carotid artery. The angiographic mean luminary diameters of the group treated with 2 and 4 μM sulforaphane were 0.25±0.1 and 0.09±0.1 mm², respectively, while the value of the control groups was 0.40±0.1 mm², indicating that sulforaphane may inhibit neointimal formation. The expression of PCNA, maker for cell cycle arrest, was decreased, while that of p53 and p21 was increased, which showed the same pattern as one in in-vitro study. These results suggest that sulforaphane-inhibited VSMC proliferation may occur through the G1/S cell cycle arrest by up-regulation of p53 signaling pathway, and then lead to the decreased neointimal hyperplasia thickening. Thus, sulforaphane may be a promising candidate for the therapy of atherosclerosis and post-angiography restenosis. © 2013.
N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) suppresses the proliferation of PANC-1 pancreatic cancer cells via apoptosis and G2/M cell cycle arrest.

PubMed

Chen, Su-Feng; Xia, Jun; Lv, Ya-Ping; Liu, Jin-Lin; Li, Wan-Xiang; Yu, Xi-Ping; Hu, Wei-Xiao; Zhou, Yong-Lie

2015-04-01

Pancreatic cancer is one of the human gastrointestinal malignancies with a high mortality and poor prognosis. Approximately eighty percent of patients are diagnosed with unresectable or metastatic disease. Thus, development of novel chemicals in the treatment of pancreatic cancer is imperative. This study aimed to investigate the anticancer effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a new tetrazine derivative, on the PANC-1 pancreatic cancer cell line and clarify the underlying molecular mechanism. Using an MTT assay, we found that ZGDHu-1 significantly suppressed the proliferation of PANC-1 cells in a time- and dose-dependent manner. Moreover, according to the morphological and flow cytometric analysis, the results indicated that ZGDHu-1 induced PANC-1 cell apoptosis and G2/M cell cycle arrest in a dose-dependent manner. In the western blot analysis, expression of the pro-apoptotic Bax gene was upregulated while the anti-apoptotic Bcl-2 gene was downregulated following treatment with ZGDHu-1. ZGDHu-1 also activated pro-caspase-3 and PARP and increased the expression of NF-κB inhibitor IκB. Furthermore, the expression levels of G2/M regulatory molecules such as cyclin B1 and cdc2 were decreased while that of Chk1 was increased. These results suggested that ZGDHu-1 suppressed the proliferation of pancreatic cancer cells, rendering it a potential therapeutic drug for the treatment of pancreatic cancer.
Involvement of prolyl isomerase PIN1 in the cell cycle progression and proliferation of hepatic oval cells.

PubMed

Risal, Prabodh; Shrestha, Nirajan; Chand, Lokendra; Sylvester, Karl G; Jeong, Yeon Jun

2017-04-01

Liver regenerates remarkably after toxic injury or surgical resection. In the case of failure of resident hepatocytes to restore loss, repopulation is carried out by induction, proliferation, and differentiation of the progenitor cell. Although, some signaling pathways have been verified to contribute oval cell-mediated liver regeneration, role of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin1) in the oval cells proliferation is unknown. In the present study, we evaluate the role of Pin1 in oval cells proliferation. In our study, the expression of Pin1 in the mice liver increased after three weeks feeding of 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) diet along with the proliferation of oval cells. The expression of Pin1 was higher in oval cells compared to the hepatocytes.Pin1 inhibition by Juglone reduced oval cell proliferation, which was restored to normal when oval cells were treated with IGF-1. Consistent with increased cell growth, expression of Pin1, β-catenin and PCNA were increased in IGF-1 treated cells in a time dependent manner. In FACS analysis, siRNA-mediated knockdown of the Pin1 protein in the oval cells significantly increased the numbers of cells in G0/G1 phase. Furthermore, hepatocyte when treated with TGF-β showed marked reduction in cell proliferation and expression of Pin1 whereas this effect was not seen in the oval cells treated with TGF-β. In conclusion, Pin1 plays important role in the cell cycle progression and increase oval cells proliferation which may be crucial in chronic liver injury. Copyright © 2017 Elsevier GmbH. All rights reserved.
FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity.

PubMed

Delpoux, Arnaud; Lai, Chen-Yen; Hedrick, Stephen M; Doedens, Andrew L

2017-10-17

The factors and steps controlling postinfection CD8 + T cell terminal effector versus memory differentiation are incompletely understood. Whereas we found that naive TCF7 (alias "Tcf-1") expression is FOXO1 independent, early postinfection we report bimodal, FOXO1-dependent expression of the memory-essential transcription factor TCF7 in pathogen-specific CD8 + T cells. We determined the early postinfection TCF7 high population is marked by low TIM3 expression and bears memory signature hallmarks before the appearance of established memory precursor marker CD127 (IL-7R). These cells exhibit diminished TBET, GZMB, mTOR signaling, and cell cycle progression. Day 5 postinfection, TCF7 high cells express higher memory-associated BCL2 and EOMES, as well as increased accumulation potential and capacity to differentiate into memory phenotype cells. TCF7 retroviral transduction opposes GZMB expression and the formation of KLRG1 pos phenotype cells, demonstrating an active role for TCF7 in extinguishing the effector program and forestalling terminal differentiation. Past the peak of the cellular immune response, we report a gradient of FOXO1 and TCF7 expression, which functions to oppose TBET and orchestrate a continuum of effector-to-memory phenotypes.
Suppression of IL-1beta-induced COX-2 expression by trichostatin A (TSA) in human endometrial stromal cells.

PubMed

Wu, Yan; Guo, Sun-Wei

2007-11-01

Over-production of cyclooxygenase-2 (COX-2) plays an important role in the positive feedback loop that leads to proliferation and inflammation in endometriosis. Following our observation that histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and valproic acid (VPA) can suppress proliferation of endometrial stromal cells, we sought to determine whether TSA suppresses IL-1beta-induced COX-2 expression in endometrial stromal cells. In vitro study using a recently established immortalized endometrial stromal cell line. The stromal cells were pretreated with TSA before stimulation with IL-1beta, and COX-2 gene and protein expression was measured by real-time quantitative RT-PCR and Western blot analysis, respectively. IL-1beta stimulated COX-2 expression in a concentration-dependent manner in endometrial stromal cells. The induced COX-2 gene and protein expression were suppressed by TSA pretreatment. TSA suppresses IL-1beta-induced COX-2 gene and protein expression in endometrial stromal cells. This finding, coupled with the findings that TSA and another HDACI, valproic acid, suppress proliferation and induce cell cycle arrest, suggests that HDACIs are a promising class of compound that has therapeutic potential for endometriosis.
Regulatory network analysis of Epstein-Barr virus identifies functional modules and hub genes involved in infectious mononucleosis.

PubMed

Poorebrahim, Mansour; Salarian, Ali; Najafi, Saeideh; Abazari, Mohammad Foad; Aleagha, Maryam Nouri; Dadras, Mohammad Nasr; Jazayeri, Seyed Mohammad; Ataei, Atousa; Poortahmasebi, Vahdat

2017-05-01

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis (IM) and establishes lifetime infection associated with a variety of cancers and autoimmune diseases. The aim of this study was to develop an integrative gene regulatory network (GRN) approach and overlying gene expression data to identify the representative subnetworks for IM and EBV latent infection (LI). After identifying differentially expressed genes (DEGs) in both IM and LI gene expression profiles, functional annotations were applied using gene ontology (GO) and BiNGO tools, and construction of GRNs, topological analysis and identification of modules were carried out using several plugins of Cytoscape. In parallel, a human-EBV GRN was generated using the Hu-Vir database for further analyses. Our analysis revealed that the majority of DEGs in both IM and LI were involved in cell-cycle and DNA repair processes. However, these genes showed a significant negative correlation in the IM and LI states. Furthermore, cyclin-dependent kinase 2 (CDK2) - a hub gene with the highest centrality score - appeared to be the key player in cell cycle regulation in IM disease. The most significant functional modules in the IM and LI states were involved in the regulation of the cell cycle and apoptosis, respectively. Human-EBV network analysis revealed several direct targets of EBV proteins during IM disease. Our study provides an important first report on the response to IM/LI EBV infection in humans. An important aspect of our data was the upregulation of genes associated with cell cycle progression and proliferation.
Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiaro, Christopher, E-mail: cchiaro@tcmedc.org; Lazarova, Darina L., E-mail: dlazarova@tcmedc.org; Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org

2012-11-09

Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116,more » does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.« less
Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

PubMed Central

Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

2010-01-01

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991
Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

PubMed

Yothaisong, Supak; Dokduang, Hasaya; Anzai, Naohiko; Hayashi, Keitaro; Namwat, Nisana; Yongvanit, Puangrat; Sangkhamanon, Sakkarn; Jutabha, Promsuk; Endou, Hitoshi; Loilome, Watcharin

2017-03-01

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na + -independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.
Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

PubMed

Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

2015-01-01

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.
Biochemical effects of veterinary antibiotics on proliferation and cell cycle arrest of human HEK293 cells.

PubMed

Kim, Hyeon Young; Kim, Ki-Tae; Kim, Sang Don

2012-08-01

The purpose of this study was to examine the effects of veterinary antibiotics, including amoxicillin (AMX), chlortetracycline (CTC) and tylosin (TYL), on the biochemical mechanism of human embryonic kidney cells (HEK293). CTC and TYL inhibited HEK293 cell proliferation, in both time- and dose-dependent manners, and changed the cell morphology; whereas, AMX showed no cytotoxic effects. The cell cycle analysis of CTC and TYL revealed G1-arrest in HEK293 cells. Western blot analysis also showed that CTC and TYL affected the activation of DNA damage responsive proteins, as well as cell cycle regulatory proteins, such as p53, p21(Waf1/Cip1) and Rb protein, which are crucial in the G1-S transition. The activation of p21(Waf1/Cip1) was significantly up-regulated over time, but there was no change in the level of CDK2 expression. The results of this study suggest that veterinary antibiotics, even at low level concentrations on continuous exposure, can potentially risk the development of human cells.
qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells.

PubMed

Andrews, J O; Conway, W; Cho, W -K; Narayanan, A; Spille, J -H; Jayanth, N; Inoue, T; Mullen, S; Thaler, J; Cissé, I I

2018-05-09

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.

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