The zebrafish maternal-effect gene cellular atoll encodes the centriolar component sas-6 and defects in its paternal function promote whole genome duplication.

PubMed

Yabe, Taijiro; Ge, Xiaoyan; Pelegri, Francisco

2007-12-01

A female-sterile zebrafish maternal-effect mutation in cellular atoll (cea) results in defects in the initiation of cell division starting at the second cell division cycle. This phenomenon is caused by defects in centrosome duplication, which in turn affect the formation of a bipolar spindle. We show that cea encodes the centriolar coiled-coil protein Sas-6, and that zebrafish Cea/Sas-6 protein localizes to centrosomes. cea also has a genetic paternal contribution, which when mutated results in an arrested first cell division followed by normal cleavage. Our data supports the idea that, in zebrafish, paternally inherited centrosomes are required for the first cell division while maternally derived factors are required for centrosomal duplication and cell divisions in subsequent cell cycles. DNA synthesis ensues in the absence of centrosome duplication, and the one-cycle delay in the first cell division caused by cea mutant sperm leads to whole genome duplication. We discuss the potential implications of these findings with regards to the origin of polyploidization in animal species. In addition, the uncoupling of developmental time and cell division count caused by the cea mutation suggests the presence of a time window, normally corresponding to the first two cell cycles, which is permissive for germ plasm recruitment.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli.

PubMed

Bhat, Supriya V; Kamencic, Belma; Körnig, André; Shahina, Zinnat; Dahms, Tanya E S

2018-01-01

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro , and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force - laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

PubMed

Orth, James D; Kohler, Rainer H; Foijer, Floris; Sorger, Peter K; Weissleder, Ralph; Mitchison, Timothy J

2011-07-01

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. ©2011 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.

Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei

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

Li Ziyin; Tu Xiaoming; Wang, Ching C.

2006-11-01

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrestedmore » in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.« less

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli

PubMed Central

Bhat, Supriya V.; Kamencic, Belma; Körnig, André; Shahina, Zinnat; Dahms, Tanya E. S.

2018-01-01

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage. PMID:29472899

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

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

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs.

PubMed

Ke, Ching-Shiun; Liu, Hsiao-Sheng; Yen, Cheng-Hsin; Huang, Guan-Cheng; Cheng, Hung-Chi; Huang, Chi-Ying F; Su, Chun-Li

2014-05-01

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA-IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

TfVPS32 Regulates Cell Division in the Parasite Tritrichomonas foetus.

PubMed

Iriarte, Lucrecia S; Midlej, Victor; Frontera, Lorena S; Moros Duarte, Daniel; Barbeito, Claudio G; de Souza, Wanderley; Benchimol, Marlene; de Miguel, Natalia; Coceres, Veronica M

2018-01-01

The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Cell cycle arrest and the evolution of chronic kidney disease from acute kidney injury.

PubMed

Canaud, Guillaume; Bonventre, Joseph V

2015-04-01

For several decades, acute kidney injury (AKI) was generally considered a reversible process leading to complete kidney recovery if the individual survived the acute illness. Recent evidence from epidemiologic studies and animal models, however, have highlighted that AKI can lead to the development of fibrosis and facilitate the progression of chronic renal failure. When kidney injury is mild and baseline function is normal, the repair process can be adaptive with few long-term consequences. When the injury is more severe, repeated, or to a kidney with underlying disease, the repair can be maladaptive and epithelial cell cycle arrest may play an important role in the development of fibrosis. Indeed, during the maladaptive repair after a renal insult, many tubular cells that are undergoing cell division spend a prolonged period in the G2/M phase of the cell cycle. These tubular cells recruit intracellular pathways leading to the synthesis and the secretion of profibrotic factors, which then act in a paracrine fashion on interstitial pericytes/fibroblasts to accelerate proliferation of these cells and production of interstitial matrix. Thus, the tubule cells assume a senescent secretory phenotype. Characteristic features of these cells may represent new biomarkers of fibrosis progression and the G2/M-arrested cells may represent a new therapeutic target to prevent, delay or arrest progression of chronic kidney disease. Here, we summarize recent advances in our understanding of the biology of the cell cycle and how cell cycle arrest links AKI to chronic kidney disease. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

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.

Arrested embryonic development: a review of strategies to delay hatching in egg-laying reptiles

PubMed Central

Rafferty, Anthony R.; Reina, Richard D.

2012-01-01

Arrested embryonic development involves the downregulation or cessation of active cell division and metabolic activity, and the capability of an animal to arrest embryonic development results in temporal plasticity of the duration of embryonic period. Arrested embryonic development is an important reproductive strategy for egg-laying animals that provide no parental care after oviposition. In this review, we discuss each type of embryonic developmental arrest used by oviparous reptiles. Environmental pressures that might have directed the evolution of arrest are addressed and we present previously undiscussed environmentally dependent physiological processes that may occur in the egg to bring about arrest. Areas for future research are proposed to clarify how ecology affects the phenotype of developing embryos. We hypothesize that oviparous reptilian mothers are capable of providing their embryos with a level of phenotypic adaptation to local environmental conditions by incorporating maternal factors into the internal environment of the egg that result in different levels of developmental sensitivity to environmental conditions after they are laid. PMID:22438503

Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

PubMed

Cesare, Anthony J

2014-11-01

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

PubMed

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Cyclophilin A Levels Dictate Infection Efficiency of Human Immunodeficiency Virus Type 1 Capsid Escape Mutants A92E and G94D ▿

PubMed Central

Ylinen, Laura M. J.; Schaller, Torsten; Price, Amanda; Fletcher, Adam J.; Noursadeghi, Mahdad; James, Leo C.; Towers, Greg J.

2009-01-01

Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration. PMID:19073742

Citral induces auxin and ethylene-mediated malformations and arrests cell division in Arabidopsis thaliana roots.

PubMed

Graña, E; Sotelo, T; Díaz-Tielas, C; Araniti, F; Krasuska, U; Bogatek, R; Reigosa, M J; Sánchez-Moreiras, A M

2013-02-01

Citral is a linear monoterpene which is present, as a volatile component, in the essential oil of several different aromatic plants. Previous studies have demonstrated the ability of citral to alter the mitotic microtubules of plant cells, especially at low concentrations. The changes to the microtubules may be due to the compound acting directly on the treated root and coleoptile cells or to indirect action through certain phytohormones. This study, performed in Arabidopsis thaliana, analysed the short-term effects of citral on the auxin content and mitotic cells, and the long-term effects of these alterations on root development and ethylene levels. The results of this study show that citral alters auxin content and cell division and has a strong long-term disorganising effect on cell ultra-structure in A. thaliana seedlings. Its effects on cell division, the thickening of the cell wall, the reduction in intercellular communication, and the absence of root hairs confirm that citral is a strong phytotoxic compound, which has persistent effects on root development.

Karyological observations

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; O'Connor, S. A.

1984-01-01

Root tips prepared for metaphase chromosome analysis from seedlings germinated under microgravity on the Space Shuttle (oats and mung bean) or which were exposed to space flight as very young seedlings (sunflower) have been examined. Experimental constraints did not permit pre-fixation in space with a cytostatic agent but arrest was achieved in the first division cycle on Earth after recovery. The number of cells in division was significantly depressed in all three species. Several chromosomal abnormalities were encountered in flight material. Bridge formation was seen in sunflower, as was aneuploidy. Breakage and fracture of chromosomes was prevalent in oats. No aberrant features could be detected in the chromosomes of mung bean. These results, although preliminary, should serve to alert investigators of the need to assess carefully as many aspects of cell division in higher plants exposed to space flight conditions as possible.

Natural Compounds as Modulators of Cell Cycle Arrest: Application for Anticancer Chemotherapies

PubMed Central

Bailon-Moscoso, Natalia; Cevallos-Solorzano, Gabriela; Romero-Benavides, Juan Carlos; Orellana, Maria Isabel Ramirez

2017-01-01

Natural compounds from various plants, microorganisms and marine species play an important role in the discovery novel components that can be successfully used in numerous biomedical applications, including anticancer therapeutics. Since uncontrolled and rapid cell division is a hallmark of cancer, unraveling the molecular mechanisms underlying mitosis is key to understanding how various natural compounds might function as inhibitors of cell cycle progression. A number of natural compounds that inhibit the cell cycle arrest have proven effective for killing cancer cells in vitro, in vivo and in clinical settings. Significant advances that have been recently made in the understanding of molecular mechanisms underlying the cell cycle regulation using the chemotherapeutic agents is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to anticancer drugs, especially of natural origin, which inhibit the activities of cyclins and cyclin-dependent kinases, as well as other proteins and enzymes involved in proper regulation of cell cycle leading to controlled cell proliferation. PMID:28367072

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

PubMed Central

Roth, Therese M.; Chiang, C.-Y. Ason; Inaba, Mayu; Yuan, Hebao; Salzmann, Viktoria; Roth, Caitlin E.; Yamashita, Yukiko M.

2012-01-01

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions. PMID:22357619

Cell cycle in egg cell and its progression during zygotic development in rice.

PubMed

Sukawa, Yumiko; Okamoto, Takashi

2018-03-01

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

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

Analysis of genomic instability in primary spermatocytes of interspecific hybrids of the red fox (Vulpes vulpes) and the Arctic fox (Alopex lagopus).

PubMed

Bugno-Poniewierska, Monika; Pawlina, Klaudia; Jakubczak, Andrzej; Jeżewska-Witkowska, Grażyna

2014-01-01

The aim of this study was to analyse meiotic cells of male interspecific hybrids of the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus). To this end we determined stages of meiotic cells as well as carried out FISH analyses with probes specific to heterosomes and a TUNEL assay on synaptonemal complex preparations. The meiotic cell analysis revealed only the presence of stages of the first meiotic division from leptotene to pachytene. Moreover, we observed an increased level of early dissociation of the X-Y bivalent as well as a high percentage of apoptotic cells. These results indicate the disruption of meiotic division in male hybrids manifested through meiotic arrest of the cells. Faulty pairing of the heterosomes can be considered as one of the causes leading to the initiation of the apoptotic process.

v-Src-induced nuclear localization of YAP is involved in multipolar spindle formation in tetraploid cells.

PubMed

Kakae, Keiko; Ikeuchi, Masayoshi; Kuga, Takahisa; Saito, Youhei; Yamaguchi, Naoto; Nakayama, Yuji

2017-01-01

The protein-tyrosine kinase, c-Src, is involved in a variety of signaling events, including cell division. We have reported that v-Src, which is a mutant variant of the cellular proto-oncogene, c-Src, causes delocalization of Aurora B kinase, resulting in a furrow regression in cytokinesis and the generation of multinucleated cells. However, the effect of v-Src on mitotic spindle formation is unknown. Here we show that v-Src-expressing HCT116 and NIH3T3 cells undergo abnormal cell division, in which cells separate into more than two cells. Upon v-Src expression, the proportion of multinucleated cells is increased in a time-dependent manner. Flow cytometry analysis revealed that v-Src increases the number of cells having a ≥4N DNA content. Microscopic analysis showed that v-Src induces the formation of multipolar spindles with excess centrosomes. These results suggest that v-Src induces multipolar spindle formation by generating multinucleated cells. Tetraploidy activates the tetraploidy checkpoint, leading to a cell cycle arrest of tetraploid cells at the G1 phase, in which the nuclear exclusion of the transcription co-activator YAP plays a critical role. In multinucleated cells that are induced by cytochalasin B and the Plk1 inhibitor, YAP is excluded from the nucleus. However, v-Src prevents this nuclear exclusion of YAP through a decrease in the phosphorylation of YAP at Ser127 in multinucleated cells. Furthermore, v-Src decreases the expression level of p53, which also plays a critical role in the cell cycle arrest of tetraploid cells. These results suggest that v-Src promotes abnormal spindle formation in at least two ways: generation of multinucleated cells and a weakening of the tetraploidy checkpoint. Copyright © 2016 Elsevier Inc. All rights reserved.

Specific biomarkers for stochastic division patterns and starvation-induced quiescence under limited glucose levels in fission yeast

PubMed Central

Pluskal, Tomáš; Hayashi, Takeshi; Saitoh, Shigeaki; Fujisawa, Asuka; Yanagida, Mitsuhiro

2011-01-01

Glucose as a source of energy is centrally important to our understanding of life. We investigated the cell division–quiescence behavior of the fission yeast Schizosaccharomyces pombe under a wide range of glucose concentrations (0–111 mm). The mode of S. pombe cell division under a microfluidic perfusion system was surprisingly normal under highly diluted glucose concentrations (5.6 mm, 1/20 of the standard medium, within human blood sugar levels). Division became stochastic, accompanied by a curious division-timing inheritance, in 2.2–4.4 mm glucose. A critical transition from division to quiescence occurred within a narrow range of concentrations (2.2–1.7 mm). Under starvation (1.1 mm) conditions, cells were mostly quiescent and only a small population of cells divided. Under fasting (0 mm) conditions, division was immediately arrested with a short chronological lifespan (16 h). When cells were first glucose starved prior to fasting, they possessed a substantially extended lifespan (∼14 days). We employed a quantitative metabolomic approach for S. pombe cell extracts, and identified specific metabolites (e.g. biotin, trehalose, ergothioneine, S-adenosyl methionine and CDP-choline), which increased or decreased at different glucose concentrations, whereas nucleotide triphosphates, such as ATP, maintained high concentrations even under starvation. Under starvation, the level of S-adenosyl methionine increased sharply, accompanied by an increase in methylated amino acids and nucleotides. Under fasting, cells rapidly lost antioxidant and energy compounds, such as glutathione and ATP, but, in fasting cells after starvation, these and other metabolites ensuring longevity remained abundant. Glucose-starved cells became resistant to 40 mm H2O2 as a result of the accumulation of antioxidant compounds. PMID:21306563

Identifying Developmental Zones in Maize Lateral Root Cell Length Profiles using Multiple Change-Point Models

PubMed Central

Moreno-Ortega, Beatriz; Fort, Guillaume; Muller, Bertrand; Guédon, Yann

2017-01-01

The identification of the limits between the cell division, elongation and mature zones in the root apex is still a matter of controversy when methods based on cellular features, molecular markers or kinematics are compared while methods based on cell length profiles have been comparatively underexplored. Segmentation models were developed to identify developmental zones within a root apex on the basis of epidermal cell length profiles. Heteroscedastic piecewise linear models were estimated for maize lateral roots of various lengths of both wild type and two mutants affected in auxin signaling (rtcs and rum-1). The outputs of these individual root analyses combined with morphological features (first root hair position and root diameter) were then globally analyzed using principal component analysis. Three zones corresponding to the division zone, the elongation zone and the mature zone were identified in most lateral roots while division zone and sometimes elongation zone were missing in arrested roots. Our results are consistent with an auxin-dependent coordination between cell flux, cell elongation and cell differentiation. The proposed segmentation models could extend our knowledge of developmental regulations in longitudinally organized plant organs such as roots, monocot leaves or internodes. PMID:29123533

Drosophila variable nurse cells encodes Arrest defective 1 (ARD1), the catalytic subunit of the major N-terminal acetyltransferase complex

PubMed Central

Wang, Ying; Mijares, Michelle; Gall, Megan D.; Turan, Tolga; Javier, Anna; Bornemann, Douglas J; Manage, Kevin; Warrior, Rahul

2010-01-01

Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Further we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes. PMID:20882681

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

PubMed Central

Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

2011-01-01

Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, Spindle Assembly Checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of micronuclei-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein. PMID:21412778

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski.

PubMed

Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

2012-01-01

Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski-/- mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of MN-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein. Copyright © 2011 Wiley Periodicals, Inc.

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

p53 protects against genome instability following centriole duplication failure

PubMed Central

Lambrus, Bramwell G.; Uetake, Yumi; Clutario, Kevin M.; Daggubati, Vikas; Snyder, Michael; Sluder, Greenfield

2015-01-01

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure. PMID:26150389

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

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

Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplicationmore » in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.« less

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

PubMed

Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

2014-03-04

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes

PubMed Central

Shenje, Lincoln T.; Andersen, Peter; Halushka, Marc K.; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B.; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A. S.; Chen, Yan; Chelko, Stephen; Crosson, Jane E.; Scheel, Janet; Vricella, Luca; Craig, Brian D.; Marosy, Beth A.; Mohr, David W.; Hetrick, Kurt N.; Romm, Jane M.; Scott, Alan F.; Valle, David; Naggert, Jürgen K.; Kwon, Chulan; Doheny, Kimberly F.; Judge, Daniel P.

2014-01-01

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest. PMID:24595103

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells

PubMed Central

Zhang, Yu; Xue, Ying-bo; Li, Hang; Qiu, Dong; Wang, Zhi-wei; Tan, Shi-sheng

2017-01-01

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients. PMID:28165402

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells.

PubMed

Zhang, Yu; Xue, Ying-Bo; Li, Hang; Qiu, Dong; Wang, Zhi-Wei; Tan, Shi-Sheng

2017-02-04

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae.

PubMed

Yamamoto, Daisuke S; Tachibana, Kazunori; Sumitani, Megumi; Lee, Jae Min; Hatakeyama, Masatsugu

2008-01-01

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.

Senescence and the pro-tumorigenic stroma.

PubMed

Alspach, Elise; Fu, Yujie; Stewart, Sheila A

2013-01-01

Hayflick and Moorhead first described senescence in the late 1960's as a permanent growth arrest that primary cells underwent after a defined number of cellular divisions in culture. This observation gave rise to the hypothesis that cells contained an internal counting mechanism that limited cellular division and that this limit was an important barrier to cellular transformation. What began as an in vitro observation has led to an immense body of work that reaches into all fields of biology and is of particular interest in the areas of aging, tissue regeneration, and tumorigenesis. The initially simplistic view that senescence limits cellular division and contributes to aging while stymying tumorigenesis has now evolved into an important and complex biological process that has numerous caveats and often opposing effects on tumorigenesis. In this review, we limit our discussion to the complex role senescence plays in tumorigenesis. Throughout the review we attempt to draw many parallels to other systems including the role senescent cells play in the tumor microenvironment and their significant molecular and phenotypic similarities to cancer associated fibroblasts (CAFs).

Collective and single cell behavior in epithelial contact inhibition.

PubMed

Puliafito, Alberto; Hufnagel, Lars; Neveu, Pierre; Streichan, Sebastian; Sigal, Alex; Fygenson, D Kuchnir; Shraiman, Boris I

2012-01-17

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

PubMed

Abbouni, Bouziane; Elhariry, Hesham M; Auling, Georg

2003-01-01

Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

Cell cycle in ascidian eggs and embryos.

PubMed

McDougall, Alex; Chenevert, Janet; Lee, Karen W; Hebras, Celine; Dumollard, Remi

2011-01-01

In ascidians the cell cycle machinery has been studied mainly in oocytes while ascidian embryos have been used to dissect the mechanism that controls asymmetric cell division (ACD). Here we overview the most specific and often exceptional points and events in cell cycle control in ascidian oocytes and early embryos. Mature stage IV eggs are arrested at metaphase I due to cytostatic factor (CSF). In vertebrates, unfertilized eggs are arrested at metaphase II by CSF. Meta II-CSF is mediated by the Mos/MEK/MAPK/Erp1 pathway, which inhibits the ubiquitin ligase APC/C(cdc20) preventing cyclin B destruction thus stabilizing MPF activity. CSF is inactivated by the fertilization Ca(2+) transient that stimulates the destruction of Erp1 thus releasing APC/C(cdc20) from inhibition. Although many of the components of CSF are conserved between the ascidian and the vertebrates, the lack of Erp1 in the ascidians (and indeed other invertebrates) is notable since the Mos/MAPK pathway nonetheless mediates Meta I-CSF. Moreover, since the fertilization Ca(2+) transient targets Erp1, it is not clear how the sperm-triggered Ca(2+) transient in ascidians (and again other invertebrates) stimulates cyclin B destruction in the absence of Erp1. Nonetheless, like mammalian eggs, sperm trigger a series of Ca(2+) oscillations that increases the rate of cyclin B destruction and the subsequent loss of MAPK activity leading to meiotic exit in ascidians. Positive feedback from MPF maintains the Ca(2+) oscillations in fertilized ascidian eggs ensuring the eventual loss of MPF stimulating the egg-to-embryo transition. Embryonic cell cycles in the ascidian are highly stereotyped where both the rate of cell division and the orientation of cell division planes are precisely controlled. Three successive rounds of ACD generate two small posterior germ cell precursors at the 64 cell stage. The centrosome-attracting body (CAB) is a macroscopic cortical structure visible by light microscopy that causes these three rounds of ACD. Entry into mitosis activates the CAB causing the whole mitotic spindle to rotate and migrate toward the cortical CAB leading to a highly ACD whereby one small cell is formed that inherits the CAB and approximately 40 maternal postplasmic/PEM RNAs including the germ cell marker vasa.

New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2

PubMed Central

Zhao, Feng; Wang, Yongtao; An, Haoran; Hu, Xiaosong

2016-01-01

ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. PMID:27578754

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents.

PubMed

Rodríguez, E M; Parra, M T; Rufas, J S; Suja, J A

2001-12-01

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Genes involved in immortalization of human mammary cells

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

Stampfer, Martha R.; Yaswen, Paul

2001-09-27

Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings ofmore » this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of enforcing a Raf-induced growth arrest and that this tumor suppressive function is lost at a specific stage in malignant transformation. Further studies to elucidate the ways by which immortal, converted HMEC escape this arrest may provide a more complete model of breast carcinogenesis as well as ways to intervene in that process.« less

Bisphenol A alters oocyte maturation by prematurely closing gap junctions in the cumulus cell-oocyte complex.

PubMed

Acuña-Hernández, Deyanira Guadalupe; Arreola-Mendoza, Laura; Santacruz-Márquez, Ramsés; García-Zepeda, Sihomara Patricia; Parra-Forero, Lyda Yuliana; Olivares-Reyes, Jesús Alberto; Hernández-Ochoa, Isabel

2018-04-01

In ovarian follicles, cumulus cells communicate with the oocyte through gap junction intercellular communication (GJIC), to nurture the oocyte and control its meiosis arrest and division. Bisphenol A (BPA) is a monomer found in polycarbonate-made containers that can induce functional alterations, including impaired oocyte meiotic division and reduced molecule transfer in GJIC. However, how BPA alters oocyte meiotic division is unclear. We investigated whether BPA effects on oocyte meiotic division were correlated with reduced transfer in GJIC. Cumulus cell-oocyte complexes (COCs) isolated from mouse preovulatory follicles were cultured with 0, 0.22, 2.2, 22, 220, and 2200 nM BPA for 2 h. An additional 16-h incubation with epidermal growth factor (EGF) was performed to promote the occurrence of meiotic resumption and progression to metaphase II. Without EGF stimulus, BPA treatment increased the percentage of oocytes undergoing meiotic resumption, decreased GJIC in the COCs, and did not modify GJIC gene (Cx43 and Cx37) and protein (CX43) expression. Following EGF stimulus, BPA increased the percentage of oocytes that remained at the anaphase and telophase stages, and decreased the percentage of oocytes reaching the metaphase II stage. Concomitantly, BPA reduced the expansion of cumulus cells. Carbenoxolone (a GJIC inhibitor) and 6-diazo-5-oxo-l-norleucine (a cumulus cell-expansion inhibitor) exerted effects on meiotic division similar to those exerted by BPA. These data suggest that BPA accelerates meiotic progression, leading to impaired prophase I-to-metaphase II transition, and that this adverse effect is correlated with reduced bidirectional communication in the COC. Copyright © 2018 Elsevier Inc. All rights reserved.

Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study.

PubMed

Ebner, T; Oppelt, P; Wöber, M; Staples, P; Mayer, R B; Sonnleitner, U; Bulfon-Vogl, S; Gruber, I; Haid, A E; Shebl, O

2015-01-01

Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest? Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth. Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca(2+) chelator could be restored by means of ionophores in an animal model. This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent. Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (≤15%). Severe male factor patients and patients with <30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, in vitro culture was performed as in the control cycles, up to blastocyst stage when achievable. Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (P < 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (P < 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (P < 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy. The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos. This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest. No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

PubMed

Kaminaga, Kiichi; Noguchi, Miho; Narita, Ayumi; Hattori, Yuya; Usami, Noriko; Yokoya, Akinari

2016-11-01

To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila.

PubMed

Kizhedathu, Amrutha; Bagul, Archit V; Guha, Arjun

2018-04-16

Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. Here we investigate the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). We report that tracheoblasts pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). We find that mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. We propose that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth. © 2018, Kizhedathu et al.

The effect of automobile exhaust particulates on cell viability, plating efficiency and cell division of mammalian tissue culture cells.

PubMed

Seemayer, N H; Hadnagy, W; Tomingas, R

1987-03-01

Extract of particulate matter (EPM) of gasoline engine exhaust induced only a slight loss of cell viability of mouse macrophages (line IC-21) in vitro, while a strong dose-dependent reduction of plating efficiency of human cell line A-549 and of Syrian hamster line 14-1b occurred. Cytological investigations of exposed macrophages of line IC-21 revealed an increase in the mitotic index from 1.5% of control values up to 14.6% at the highest tested concentration of EPM. Mitotic arrest is based almost exclusively on C-type mitoses occurring dose-dependently in the presence of EPM. Results indicate disturbances of the spindle apparatus in the presence of EPM.

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans

PubMed Central

van Rijnberk, Lotte M.; van der Horst, Suzanne E. M.; van den Heuvel, Sander; Ruijtenberg, Suzan

2017-01-01

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. PMID:28158315

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

PubMed Central

Gupta, Prabuddha; Martin, René; Knölker, Hans-Joachim; Nihalani, Deepak; Kumar Sinha, Deepak

2017-01-01

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. PMID:28678859

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

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

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-07-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed Central

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-01-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity. PMID:9649516

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

PubMed

Hu, Xiangjing; Shen, Bin; Liao, Shangying; Ning, Yan; Ma, Longfei; Chen, Jian; Lin, Xiwen; Zhang, Daoqin; Li, Zhen; Zheng, Chunwei; Feng, Yanmin; Huang, Xingxu; Han, Chunsheng

2017-06-29

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks.

PubMed

Adan, Aysun; Baran, Yusuf

2015-11-01

Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated ≥2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.

The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells.

PubMed Central

Bomford, A; Isaac, J; Roberts, S; Edwards, A; Young, S; Williams, R

1986-01-01

The effect of the iron chelator, desferrioxamine, on transferrin binding, growth rates and the cell cycle was investigated in the human leukaemic cell line, K562. At all concentrations of the chelator (2-50 microM) binding of 125I-transferrin was increased by 24 h and reached a maximum at 72-96 h. Maximum binding (6-8-fold increased) occurred in cells treated with 20 microM-desferrioxamine, in contrast with control cells which, at 96 h, showed a 50% decrease over initial binding. Scatchard analysis at 4 degrees C showed that this increased binding was due to an increase in the number of receptors, as the Kd was similar in induced (1.8 nM) and control (1.5 nM) cells. After 96 h cells, cultured with 20 and 50 microM-desferrioxamine accumulated 59Fe from bovine transferrin at over twice the rate found with control cells, reflecting the increase in transferrin receptors. Although iron uptake was unimpaired by the chelator there was a dose-dependent inhibition of cell growth, with control cells completing three divisions in 96 h and those in 10 microM-desferrioxamine only two divisions. At the highest concentration (50 microM), cell division was abrogated although cell viability was maintained (85%). In contrast, DNA synthesis was not markedly affected, except at 50 microM-desferrioxamine when incorporation of [3H]thymidine was 52% of that in control cells. Flow cytometry revealed that there was a progressive accumulation of the cells in the active phases of their cycle (S, G2 + M). Desferrioxamine may increase transferrin receptors in two ways: by chelating a regulatory pool of iron within the cell, and by arresting cells in S phase when receptors are maximally expressed. PMID:3790074

Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species.

PubMed

Yao, Shuo; Brandt, Anders; Egsgaard, Helge; Gjermansen, Claes

2012-12-01

Triacylglycerols, an energy storage compound in microalgae, are known to be accumulated after nitrogen starvation of microalgae cells. Microalgae could be of importance for future biodiesel production due to their fast growth rate and high oil content. In collections of temperature sensitive mutants of Chlamydomonas reinhardtii and Chlorella vulgaris, nine out of fourty-one mutants in C. reinhardtii and eleven out of fifty-three mutants in C. vulgaris contained increased amounts of neutral lipids, predominantly as triacylglycerols. Upon temperature induced cell-cycle arrest, these mutants showed enlarged cellular volume compared with the wild type. The C. reinhardtii mutants were analyzed further and one type of mutants displayed a shift in lipid composition from polar membrane lipids to neutral lipids after a temperature up-shift, while the second type of mutants accumulated more total lipid per cell, predominantly as neutral lipids as compared with the wild type. Three C. reinhardtii mutants were analyzed further and found to be arrested after DNA synthesis but prior to cell division in the cell cycle. These mutants will be useful in order to further understand neutral lipid accumulation in microalgae and suggest possibilities for biodiesel production by specific induction of lipid accumulation in miroalgal cultures by cell-cycle inhibition. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis.

PubMed

Wang, Lina; Tu, Zhaowei; Liu, Chao; Liu, Hongbin; Kaldis, Philipp; Chen, Zijiang; Li, Wei

2018-06-01

Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Trf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to-end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis.

From stem cell to embryo without centrioles.

PubMed

Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

2007-09-04

Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

PubMed

Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

2009-06-15

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

Transcriptome analysis of PCOS arrested 2-cell embryos.

PubMed

Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

2018-06-18

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization.

PubMed

Fox, Annette; Harland, Kim L; Kedzierska, Katherine; Kelso, Anne

2018-01-01

Effector CD8 + T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8 + cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8 + T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8 + T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8 + naïve T cells (T N ) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of T N in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes , and lowered expression of type-1 cytokines, Prf1 , Gzmb, T-BET, and Prdm1 . However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of T N in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8 + central memory T cells (T CM ) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8 + T N. In summary, while activation of T N in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of T CM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.

SIRT6 inhibits colorectal cancer stem cell proliferation by targeting CDC25A

PubMed Central

Liu, Wenguang; Wu, Manwu; Du, Hechun; Shi, Xiaoliang; Zhang, Tao; Li, Jie

2018-01-01

Silent information regulator 6 (SIRT6) is broadly considered as a tumor suppressor due to its function in the suppression of oncogene expression. However, the role of SIRT6 in colorectal cancer stem cells (CSCs) remains uncharacterized. In the present study, it was demonstrated that SIRT6 expression was reduced in colorectal CSCs. Overexpression of SIRT6 in colorectal CSCs did not induce cell apoptosis. However, SIRT6 significantly inhibited cell proliferation, colony formation and induced G0/G1 phase arrest in colorectal CSCs. In addition, SIRT6 repressed the expression of cell division cycle 25A (CDC25A), an oncogenic phosphatase. Chromatin immunoprecipitation experiments indicated that SIRT6 directly bound to the CDC25A promoter and decreased the acetylation level of histone H3 lysine 9. Altogether, these data indicated that SIRT6 inhibits colorectal cancer stem cell proliferation by targeting CDC25A. PMID:29552180

Cell cycle arrest is not yet senescence, which is not just cell cycle arrest: terminology for TOR-driven aging.

PubMed

Blagosklonny, Mikhail V

2012-03-01

Cell cycle arrest is not yet senescence. When the cell cycle is arrested, an inappropriate growth-promotion converts an arrest into senescence (geroconversion). By inhibiting the growth-promoting mTOR pathway, rapamycin decelerates geroconversion of the arrested cells. And as a striking example, while causing arrest, p53 may decelerate or suppress geroconversion (in some conditions). Here I discuss the meaning of geroconversion and also the terms gerogenes, gerossuppressors, gerosuppressants, gerogenic pathways, gero-promoters, hyperfunction and feedback resistance, regenerative potential, hypertrophy and secondary atrophy, pro-gerogenic and gerogenic cells.

Disrupting Cyclin Dependent Kinase 1 in Spermatocytes Causes Late Meiotic Arrest and Infertility in Mice1

PubMed Central

Clement, Tracy M.; Inselman, Amy L.; Goulding, Eugenia H.; Willis, William D.; Eddy, Edward M.

2015-01-01

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes. PMID:26490841

Mechanism of artemisinin phytotoxicity action: induction of reactive oxygen species and cell death in lettuce seedlings.

PubMed

Yan, Zhi-Qiang; Wang, Dan-Dan; Ding, Lan; Cui, Hai-Yan; Jin, Hui; Yang, Xiao-Yan; Yang, Jian-She; Qin, Bo

2015-03-01

Artemisinin has been recognized as an allelochemical that inhibits growth of several plant species. However, its mode of action is not well clarified. In this study, the mechanism of artemisinin phytotoxicity on lettuce seedlings was investigated. Root and shoot elongation of lettuce seedlings were inhibited by artemisinin in a concentration-dependent manner. The compound effectively arrested cell division and caused loss of cell viability in root tips of lettuce. Overproduction of reactive oxygen species (ROS) was induced by artemisinin. Lipid peroxidation, proline overproduction and reduction of chlorophyll content in lettuce seedlings were found after treatments. These results suggested that artemisinin could induce ROS overproduction, which caused membrane lipids peroxidation and cell death, and impacted mitosis and physiological processes, resulting in growth inhibition of receptor plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription

PubMed Central

Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R.

2016-01-01

Throughout cell cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

Liaisons between survivin and Plk1 during cell division and cell death.

PubMed

Colnaghi, Rita; Wheatley, Sally P

2010-07-16

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser(20) by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.

Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2.

PubMed

Zhang, Shihao; Chen, Qinghua; Liu, Qingxu; Li, Yuxi; Sun, Xiufeng; Hong, Lixin; Ji, Suyuan; Liu, Chengyan; Geng, Jing; Zhang, Weiji; Lu, Zhonglei; Yin, Zhen-Yu; Zeng, Yuanyuan; Lin, Kwang-Huei; Wu, Qiao; Li, Qiyuan; Nakayama, Keiko; Nakayama, Keiich I; Deng, Xianming; Johnson, Randy L; Zhu, Liang; Gao, Daming; Chen, Lanfen; Zhou, Dawang

2017-05-08

Polyploidy can lead to aneuploidy and tumorigenesis. Here, we report that the Hippo pathway effector Yap promotes the diploid-polyploid conversion and polyploid cell growth through the Akt-Skp2 axis. Yap strongly induces the acetyltransferase p300-mediated acetylation of the E3 ligase Skp2 via Akt signaling. Acetylated Skp2 is exclusively localized to the cytosol, which causes hyper-accumulation of the cyclin-dependent kinase inhibitor p27, leading to mitotic arrest and subsequently cell polyploidy. In addition, the pro-apoptotic factors FoxO1/3 are overly degraded by acetylated Skp2, resulting in polyploid cell division, genomic instability, and oncogenesis. Importantly, the depletion or inactivation of Akt or Skp2 abrogated Hippo signal deficiency-induced liver tumorigenesis, indicating their epistatic interaction. Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2. Copyright © 2017 Elsevier Inc. All rights reserved.

[Structural and functional organization of centromeres in plant chromosomes].

PubMed

Silkova, O G; Loginova, D B

2014-12-01

The centromere is a specific chromosomal locus that forms the protein complex and kinetochore, maintains sister chromatid cohesion, controls chromosome attachment to the spindle, and coordinates chromosome movement during mitosis and meiosis. Defective centromere assembly or its dysfunction causes cell cycle arrest, structural abnormalities of the chromosomes, and aneuploidy. This review collects the data on the structure, functions, and epigenetic modification of centromeric chromatin, the structure and functions of the kinetochore, and sister chromatid cohesion. Taken together, these data provide insight into the specific architecture and functioning of the centromere during chromosome division and segregation in plants.

Distinct mechanisms act in concert to mediate cell cycle arrest.

PubMed

Toettcher, Jared E; Loewer, Alexander; Ostheimer, Gerard J; Yaffe, Michael B; Tidor, Bruce; Lahav, Galit

2009-01-20

In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.

Role of A-Kinase anchor protein (AKAP4) in growth and survival of ovarian cancer cells.

PubMed

Kumar, Vikash; Jagadish, Nirmala; Suri, Anil

2017-08-08

Ovarian cancer represents one of the most common malignancies among women with very high mortality rate worldwide. A-kinase anchor protein 4 (AKAP4), a unique cancer testis (CT) antigen has been shown to be associated with various malignant properties of cancer cells. However, its involvement in various molecular pathways in ovarian cancer remains unknown. In present investigation, employing gene silencing approach, we examined the role of AKAP4 in cell cycle, apoptosis and epithelial-mesenchymal transition (EMT). Further, we also investigated the effect of ablation of AKAP4 on tumor growth in SCID mice ovarian cancer xenograft mouse model. Our results showed that ablation of AKAP4 resulted in increased reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest and apoptosis in ovarian cancer cells. AKAP4 knockdown lead to degradation of protien kinase A (PKA) which was rescued by proteosome inhibitor MG-132. ROS quencher N-acetyl cysteine (NAC) treatment rescued cell cycle arrest and resumed cell division. Subsequently, increased expression of pro-apoptotic molecules and decreased expression of pro-survival/anti-apoptotic factors was observed. As a result of AKAP4 depletion, DNA damage response proteins p-γH2AX, p-ATM and p21 were upregulated. Also, knockdown of CREB resulted in similar findings. Further, PKA inhibitor (H89) and oxidative stress resulted in similar phenotype of ovarian cancer cells as observed in AKAP4 ablated cells. Collectively, for the first time our data showed the involvement of AKAP4 in PKA degradation and perturbed signaling through PKA-CREB axis in AKAP4 ablated ovarian cancer cells.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Small-molecule intramimics of formin autoinhibition: a new strategy to target the cytoskeletal remodeling machinery in cancer cells.

PubMed

Lash, L Leanne; Wallar, Bradley J; Turner, Julie D; Vroegop, Steven M; Kilkuskie, Robert E; Kitchen-Goosen, Susan M; Xu, H Eric; Alberts, Arthur S

2013-11-15

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells. ©2013 AACR

Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

PubMed

De Bem, Tiago H C; Chiaratti, Marcos R; Rochetti, Raquel; Bressan, Fabiana F; Sangalli, Juliano R; Miranda, Moysés S; Pires, Pedro R L; Schwartz, Kátia R L; Sampaio, Rafael V; Fantinato-Neto, Paulo; Pimentel, José R V; Perecin, Felipe; Smith, Lawrence C; Meirelles, Flávio V; Adona, Paulo R; Leal, Cláudia L V

2011-10-01

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae.

PubMed

Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I

2014-05-27

A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae . Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific "master regulator" proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast.

Formation of solid tumors by a single multinucleated cancer cel

PubMed Central

Weihua, Zhang; Lin, Qingtang; Ramoth, Asa J.; Fan, Dominic; Fidler, Isaiah J.

2011-01-01

BACKGROUND Large multinucleated cells (MNC) commonly exist in tumorigenic cancer cell lines widely used in research, but their contributions to tumorigenesis are unknown. METHODS In this study, we characterized MNCs in the murine fibrosarcoma cell line UV-2237 in vitro and in vivo at a single cell level. RESULTS We observed that MNCs originated from a rare subpopulation of mononuclear cells; MNCs were positive for a senescent marker, β-galacosidase (SA-β-Gal); MNCs were responsible for the majority of clonogenic activity when cultured in hard agar; MNCs were more resistant to chemotherapeutic agents than were mononuclear cells; MNCs could undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo; and, most importantly a single MNC produced orthotopic subcutaneous tumors (composed mainly of mononuclear cells) that gave rise to spontaneous lung metastases in nude mice. CONCLUSIONS MNCs can be growth-arrested under stress, are highly resistant to chemotherapy, and can generate clonal orthotopic metastatic tumors PMID:21365635

A framework for evaluating developmental defects at the cellular level: An example from ten maize anther mutants using morphological and molecular data.

PubMed

Egger, Rachel L; Walbot, Virginia

2016-11-01

In seed plants, anthers are critical for sexual reproduction, because they foster both meiosis and subsequent pollen development of male germinal cells. Male-sterile mutants are analyzed to define steps in anther development. Historically the major topics in these studies are meiotic arrest and post-meiotic gametophyte failure, while relatively few studies focus on pre-meiotic defects of anther somatic cells. Utilizing morphometric analysis we demonstrate that pre-meiotic mutants can be impaired in anticlinal or periclinal cell division patterns and that final cell number in the pre-meiotic anther lobe is independent of cell number changes of individual differentiated somatic cell types. Data derived from microarrays and from cell wall NMR analyses allow us to further refine our understanding of the onset of phenotypes. Collectively the data highlight that even minor deviations from the correct spatiotemporal pattern of somatic cell proliferation can result in male sterility in Zea mays. Copyright © 2016 Elsevier Inc. All rights reserved.

Replication Stress: A Lifetime of Epigenetic Change

PubMed Central

Khurana, Simran; Oberdoerffer, Philipp

2015-01-01

DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

Sustained vs. oscillating expressions of Ngn2, Dll1 and Hes1: a model of neural differentiation of embryonic telencephalon.

PubMed

Barton, A; Fendrik, A J

2013-07-07

Neural progenitor cells show oscillatory expression of the Notch ligand Delta-like1 (Dll1), the Notch target Hes1 and the proneural gene Neurogenin 2 (Ngn2) during embryonic development of the mammalian telencephalon. On the other hand, expression of these genes is sustained in postmitotic neurons (upregulated for Ngn2 and Dll1, down regulated for Hes1). These facts suggest that a switch from oscillatory to sustained expression of proneural and other genes is critical in neural fate decisions. Moreover, despite controversies over the role of Numb in determining the neural fate in mammals, there is evidence that inheritance of Numb during neurogenic cell division is involved in neural differentiation. It is also known that mNumb activates Notch1 receptor degradation. The arrest of oscillations in a given cell may be due to increasing degradation of Notch1 brought about by mNumb during neurogenic division. We introduce a modification in a previous model of the gene network for two cells coupled by the Delta-Notch pathway (Wang et al., 2011). We analyze the consequences of an asymmetry between two neighbor cells in the rate of degradation of Notch (mimicking the effect of asymmetric inheritance of mNumb during the neurogenic division). The results show that a slight difference in Notch degradation between the two cells keeps oscillation going in one of them while oscillation stops in the other. Moreover, when Delta-Notch coupling is canceled, both cells show sustained expression (upregulated levels for Ngn2 and Dll1, downregulated for Hes1). We show that the model is stable against parameter variations. Moreover, to take into account the possible influence of the environment on both cells, neighboring cells are included in a mean field approximation. Both, parameter fluctuations and effects of the environment lead to asynchronous oscillations of Hes1/Ngn2 in different progenitor cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

PATHWAYS OF GLUCOSE CATABOLISM IN BACILLUS CEREUS1

PubMed Central

Goldman, Manuel; Blumenthal, Harold J.

1964-01-01

Goldman, Manuel (The University of Michigan, Ann Arbor), and Harold J. Blumenthal. Pathways of glucose catabolism in Bacillus cereus. J. Bacteriol. 87:377–386. 1964.—Estimates by a radiorespirometric method of the pathways of glucose catabolism of resting-cell suspensions of Bacillus cereus strain terminalis indicate that the Embden-Meyerhof pathway predominates at every stage of development, including the sporogenic and germinative phases. At the filamentous, granular, forespore, and transitional stages, 98% of the glucose was catabolized by the Embden-Meyerhof pathway, and the remainder by the hexose monophosphate oxidative pathway. Estimates of the pathways in resting spore-suspensions arrested at defined stages of development indicate that 20% of the glucose was catabolized through the hexose monophosphate pathway in germinated spores, and 10% in the swollen and elongated stages of postgermination. In cells which had completed the first cell division, the figure fell to about 2%, a level similar to that found for vegetative cells at later stages of development. The key Embden-Meyerhof enzymes, hexokinase, phosphohexoisomerase, phosphofructokinase, and aldolase, as well as several other enzymes, were present at all stages of germination and postgerminative development, supporting the radioisotopic data obtained with whole cells. As indicated by the release of C14O2 from glucose-6-C14, terminal respiration of resting-cell suspensions operates maximally in vegetative cells at the granular, fore-spore, and transitional stages. There was marked inhibition of terminal respiration during the development of spores into vegetative cells. Only slight activity occurred in the earliest vegetative stages, and maximal operation developed after about ten cell divisions. Fumarase was absent in spores until sometime late in the elongation stage. At this point, a weak but definite activity appeared which increased during later stages of development so that, by the end of about the sixth cell division, fumarase had a specific activity about 80 times that observed at elongation. PMID:14151060

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis

PubMed Central

Zhang, Hongyu; Luo, Ming; Day, Robert C.; Talbot, Mark J.; Ivanova, Aneta; Ashton, Anthony R.; Chaudhury, Abed M.; Macknight, Richard C.; Hrmova, Maria; Koltunow, Anna M.

2015-01-01

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development. PMID:26105995

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

PubMed

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Differential arrest and adhesion of tumor cells and microbeads in the microvasculature

PubMed Central

Guo, Peng; Cai, Bin; Lei, Ming; Liu, Yang

2013-01-01

To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93% of rigid beads were arrested either at arteriole–capillary intersections or in capillaries. Only 3% were at the capillary–postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule–postcapillary venule intersections and in postcapillary venules. Only 12% of tumor cells were arrested at the arteriole–capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a χ2 test (p < 0.001). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis. PMID:23880911

Differential arrest and adhesion of tumor cells and microbeads in the microvasculature.

PubMed

Guo, Peng; Cai, Bin; Lei, Ming; Liu, Yang; Fu, Bingmei M

2014-06-01

To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93% of rigid beads were arrested either at arteriole-capillary intersections or in capillaries. Only 3% were at the capillary-postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule-postcapillary venule intersections and in postcapillary venules. Only 12% of tumor cells were arrested at the arteriole-capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a χ(2) test (p < 0.001). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis.

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

PubMed

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-11-24

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype

PubMed Central

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance. PMID:26503466

Recent advances in nontoxic Escherichia coli heat-labile toxin and its derivative adjuvants.

PubMed

Ma, Yongping

2016-11-01

The nontoxic heat-labile enterotoxin (LT) of Escherichia coli and the B subunit of LT (LTB) have been extensively studied as potent vaccine adjuvants. Areas covered: This review covers the area of enterotoxin based vaccine adjuvant and summarizes the development of nontoxic LT mutant (mLT) and LTB and their potency as oral, parenteral and injection adjuvants. Recent evidences indicated that the mechanism of LTB adjuvanticity was to enhance the turnover of dendritic cells (DCs) in spleen and increase DCs capacity to perform as antigen presentation cells (APCs) encountered with T cells. LTB also induces B and T cells clustering and delay/arrest in T-cell division following endocytosis or B-cell receptor (BCR) uptaking of antigen in a ganglioside-mediated manner. Expert commentary: It is pointed out that the immunogenicity of LTB (or LT) is more important than the receptor binding property (or ADP-ribosylation activity) for the adjuvanticity of LT toxoid. The immunogenicity of LTB (or LT) might confer unknown characteristics to maintain LT toxoid adjuvanticity.

Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

PubMed

Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

2018-04-01

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-proliferative effects, cell cycle G2/M phase arrest and blocking of chromosome segregation by probimane and MST-16 in human tumor cell lines

PubMed Central

Lu, Da Yong; Huang, Min; Xu, Cheng Hui; Yang, Wei Yi; Hu, Chao Xin; Lin, Li Ping; Tong, Lin Jiang; Li, Mei Hong; Lu, Wei; Zhang, Xiong Wen; Ding, Jian

2005-01-01

Background Anticancer bisdioxopiperazines, including ICRF-154, razoxane (Raz, ICRF-159) and ICRF-193, are a family of anticancer agents developed in the UK, especially targeting metastases of neoplasms. Two other bisdioxopiperazine derivatives, probimane (Pro) and MST-16, were synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. Cytotoxic activities and mechanisms of Raz (+)-steroisomer (ICRF-187, dexrazoxane), Pro and MST-16 against tumor cells were evaluated by MTT colorimetry, flow cytometry and karyotyping. Results Pro was cytotoxic to human tumor cell lines in vitro (IC50<50 μM for 48 h). Four human tumor cell lines (SCG-7901, K562, A549 and HL60) were susceptible to Pro at low inhibitory concentrations (IC50 values < 10 μM for 48 h). Although the IC50 against HeLa cell line of vincristine (VCR, 4.56 μM), doxorubicin (Dox, 1.12 μM) and 5-fluoruouracil (5-Fu, 0.232 μM) are lower than Pro (5.12 μM), ICRF-187 (129 μM) and MST-16 (26.4 μM), VCR, Dox and 5-Fu shows a low dose-related – high cytotoxic activity. Time-response studies showed that the cytotoxic effects of Pro are increased for 3 days in human tumor cells, whereas VCR, Dox and 5-Fu showed decreased cytotoxic action after 24 h. Cell cycle G2/M phase arrest and chromosome segregation blocking by Pro and MST-16 were noted. Although there was similar effects of Pro and MST-16 on chromosome segregation blocking action and cell cycle G2/M phase arrest at 1- 4 μM, cytotoxicity of Pro against tumor cells was higher than that of MST-16 in vitro by a factor of 3- 10 folds. Our data show that Pro may be more effective against lung cancer and leukemia while ICRF-187 and MST-16 shows similar IC50 values only against leukemia. Conclusion It suggests that Pro has a wider spectrum of cytotoxic effects against human tumor cells than other bisdioxopiperazines, especially against solid tumors, and with a single cytotoxic pathway of Pro and MST-16 affecting chromosome segregation and leading also to cell G2/ M phase arrests, which finally reduces cell division rates. Pro may be more potent than MST-16 in cytotoxicity. High dose- and time- responses of Pro, when compared with VCR, 5-Fu and Dox, were seen that suggest a selectivity of Pro against tumor growth. Compounds of bisdioxopiperazines family may keep up their cytotoxic effects longer than many other anticancer drugs. PMID:15963241

Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest.

PubMed

Li, Long-Zhu; Deng, Hong-Xia; Lou, Wen-Zhu; Sun, Xue-Yan; Song, Meng-Wan; Tao, Jing; Xiao, Bing-Xiu; Guo, Jun-Ming

2012-01-07

To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry. The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G₀/G₁ phase, whereas cells treated with high concentrations of PBA were arrested at the G₂/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G₀/ G₁ phase, cells treated with high concentrations of PBA were arrested at the S phase. The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G₀ /G₁ and G₂/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G₀/G₁ and S phases.

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

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis.

PubMed

Jarry, Marie; Lecointre, Céline; Malleval, Céline; Desrues, Laurence; Schouft, Marie-Thérèse; Lejoncour, Vadim; Liger, François; Lyvinec, Gildas; Joseph, Benoît; Loaëc, Nadège; Meijer, Laurent; Honnorat, Jérôme; Gandolfo, Pierrick; Castel, Hélène

2014-11-01

Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis

PubMed Central

Jarry, Marie; Lecointre, Céline; Malleval, Céline; Desrues, Laurence; Schouft, Marie-Thérèse; Lejoncour, Vadim; Liger, François; Lyvinec, Gildas; Joseph, Benoît; Loaëc, Nadège; Meijer, Laurent; Honnorat, Jérôme; Gandolfo, Pierrick; Castel, Hélène

2014-01-01

Background Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. Methods The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Results Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Conclusion Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma. PMID:24891448

Generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells.

PubMed

Shrotriya, Sangeeta; Deep, Gagan; Gu, Mallikarjuna; Kaur, Manjinder; Jain, Anil K; Inturi, Swetha; Agarwal, Rajesh; Agarwal, Chapla

2012-04-01

Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.

[10]-Gingerol, a major phenolic constituent of ginger root, induces cell cycle arrest and apoptosis in triple-negative breast cancer cells.

PubMed

Bernard, Megan M; McConnery, Jason R; Hoskin, David W

2017-04-01

The ginger rhizome is rich in bioactive compounds, including [6]-gingerol, [8]-gingerol, and [10]-gingerol; however, to date, most research on the anti-cancer activities of gingerols have focused on [6]-gingerol. In this study, we compared [10]-gingerol with [8]-gingerol and [6]-gingerol in terms of their ability to inhibit the growth of human and mouse mammary carcinoma cells. A colorimetric assay based on the enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide revealed that [10]-gingerol was more potent than [6]-gingerol and at least as potent as [8]-gingerol for the inhibition of triple-negative human (MDA-MB-231, MDA-MB-468) and mouse (4T1, E0771) mammary carcinoma cell growth. Further investigation of [10]-gingerol showed that it suppressed the growth of estrogen receptor-bearing (MCF-7, T47D) and HER2-overexpressing (SKBR3) breast cancer cells. The inhibitory effect of [10]-gingerol on the growth of MDA-MB-231 cells was associated with a reduction in the number of rounds of cell division and evidence of S phase-cell cycle arrest, as well as induction of apoptosis due to mitochondrial outer membrane permeabilization and the release of proapoptotic mitochondrial cytochrome c and SMAC/DIABLO into the cytoplasm. Surprisingly, killing of MDA-MB-231 cells by [10]-gingerol was not affected by a pan-caspase inhibitor (zVAD-fmk) or an anti-oxidant (N-acetylcysteine), suggesting that the cytotoxic effect of [10]-gingerol did not require caspase activation or the accumulation of reactive oxygen species. These findings suggest that further investigation of [10]-gingerol is warranted for its possible use in the treatment of breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells

PubMed Central

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-01

Abstract Previously, we analyzed protein abundance changes across a ‘minimally perturbed’ cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/), an online, searchable resource. DOI: http://dx.doi.org/10.7554/eLife.04534.001 PMID:25555159

Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis.

PubMed

Annibali, Daniela; Whitfield, Jonathan R; Favuzzi, Emilia; Jauset, Toni; Serrano, Erika; Cuartas, Isabel; Redondo-Campos, Sara; Folch, Gerard; Gonzàlez-Juncà, Alba; Sodir, Nicole M; Massó-Vallés, Daniel; Beaulieu, Marie-Eve; Swigart, Lamorna B; Mc Gee, Margaret M; Somma, Maria Patrizia; Nasi, Sergio; Seoane, Joan; Evan, Gerard I; Soucek, Laura

2014-08-18

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042

LONO1 Encoding a Nucleoporin Is Required for Embryogenesis and Seed Viability in Arabidopsis1[C][W][OA

PubMed Central

Braud, Christopher; Zheng, Wenguang; Xiao, Wenyan

2012-01-01

Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis. PMID:22898497

76 FR 6827 - Agency Information Collection Activities: Proposed Collection, Comments Requested

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-08

... Services Division (CJIS), Biometric Services Section, Support Services Unit, Module E-1, 1000 Custer Hollow... collection is needed to report completion of an arrest record. Acceptable data is stored as part of the...

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

Radiation Exposure Enhances Hepatocyte Proliferation in Neonatal Mice but not in Adult Mice.

PubMed

Shang, Yi; Sawa, Yurika; Blyth, Benjamin J; Tsuruoka, Chizuru; Nogawa, Hiroyuki; Shimada, Yoshiya; Kakinuma, Shizuko

2017-08-01

There is a natural tendency to expect that irradiation of an infant organ prior to development-related expansion will result in a higher risk of developing cancer than that of fully-developed adult tissue, and this has generally been observed. However, if tissues also vary in their initial responses to radiation depending on age, the interplay between tissue- and age-dependent risk would potentially be quite complex. We have previously shown opposing age-dependent induction of apoptosis for the intestinal epithelium and hematopoietic cells in mice, but such data are not yet available for the liver. Here, we have examined markers of DNA damage, initiation of DNA damage responses, cell cycle arrest, apoptosis and proliferation, as well as gene expression, in the B6C3F1 mouse liver over the hours and days after irradiation of mice at 1 or 7 weeks of age. We found that induction and resolution of radiation-induced DNA damage is not accompanied by significant changes in these cellular end points in the adult liver, while in infant hepatocytes modest induction of p53 accumulation and p21-mediated cell cycle arrest in a small fraction of damaged cells was overshadowed by a further stimulation of proliferation over the relatively high levels already found in the neonatal liver. We observed distinct expression of genes that regulate cell division between the ages, which may contribute to the differential responses. These data suggest that the growth factor signaling environment of the infant liver may mediate radiation-induced proliferation and increased liver cancer risk after irradiation during early life.

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

PubMed Central

McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

2015-01-01

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility. PMID:26491874

A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization.

PubMed

Bałakier, H; Casper, R F

1991-04-01

The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48-72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Several one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Polyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morphologically despite their lethal chromosomal abnormalities.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

XL413, a cell division cycle 7 kinase inhibitor enhanced the anti-fibrotic effect of pirfenidone on TGF-β1-stimulated C3H10T1/2 cells via Smad2/4.

PubMed

Jin, Shu-fang; Ma, Hai-long; Liu, Zhong-long; Fu, Shui-ting; Zhang, Chen-ping; He, Yue

2015-12-10

Pirfenidone is an orally bioavailable synthetic compound with therapeutic potential for idiopathic pulmonary fibrosis. It is thought to act through antioxidant and anti-fibrotic pathways. Pirfenidone inhibits proliferation and/or myofibroblast differentiation of a wide range of cell types, however, little studies have analyzed the effect of pirfenidone on the mesenchymal stem cells, which play an important role on the origin of myofibroblasts. We recently found that pirfenidone had anti-proliferative activity via G1 phase arrest and cell division cycle 7 (Cdc7) kinase expression decrease in transforming growth factor-β1 (TGF-β1)-stimulated murine mesenchymal stem C3H10T1/2 cells. Pirfenidone also had inhibiting effect on the migration and α-SMA expression. Moreover, in this study we showed for the first time that Cdc7 inhibitor XL413 enhanced the anti-fibrotic activity of pirfenidone via depressed the expression of Smad2/4 proteins, and also prevented the nuclear accumulation and translocation of Smad2 protein. In conclusion, we demonstrated that pirfenidone inhibited proliferation, migration and differentiation of TGF-β1-stimulated C3H10T1/2 cells, which could be enhanced by Cdc7 inhibitor XL413, via Smad2/4. Combination with pirfenidone and XL413 might provide a potential candidate for the treatment of TGF-β1 associated fibrosis. It needs in vivo studies to further validate its therapeutic function and safety in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis

PubMed Central

Caire-Brändli, Irène; Papadopoulos, Alexia; Malaga, Wladimir; Marais, David; Canaan, Stéphane; Thilo, Lutz

2014-01-01

During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis. PMID:24478064

Effects of caffeine on radiation-induced phenomena associated with cell- cycle traverse of mammalian cells

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

Walters, R.A.; Gurley, L.R.; Tobby, R.A.

1974-02-01

Caffeine induced a state of G/sub 1/ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following x-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cellsmore » synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The x-rayinduced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeinetreated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed. (auth)« less

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

PubMed

Wang, Juan; Huang, Shujuan; Xing, Lingxiao; Cui, Jinfeng; Tian, Ziqiang; Shen, Haitao; Jiang, Xiujuan; Yan, Xia; Wang, Junling; Zhang, Xianghong

2015-11-01

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton

PubMed Central

2017-01-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria. PMID:28945742

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

PubMed

Govindarajan, Sutharsan; Amster-Choder, Orna

2017-09-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest

PubMed Central

O’Neill, Thomas J.; Mack, Christopher P.; Taylor, Joan M.

2012-01-01

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK−/− but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium. PMID:22555221

Germinal Cell Aplasia in Kif18a Mutant Male Mice Due to Impaired Chromosome Congression and Dysregulated BubR1 and CENP-E

PubMed Central

Liu, Xue-song; Zhao, Xu-dong; Wang, Xiaoxing; Yao, Yi-xin; Zhang, Liang-liang; Shu, Run-zhe; Ren, Wei-hua; Huang, Ying; Huang, Lei; Gu, Ming-min; Kuang, Ying; Wang, Long; Lu, Shun-yuan; Chi, Jun; Fen, Jing-sheng; Wang, Yi-fei; Fei, Jian; Dai, Wei; Wang, Zhu-Gang

2010-01-01

Chromosomal instability during cell division frequently causes cell death or malignant transformation. Orderly chromosome congression at the metaphase plate, a paramount process to vertebrate mitosis and meiosis, is controlled by a number of molecular regulators, including kinesins. Kinesin-8 (Kif18A) functions to control mitotic chromosome alignment at the mid-zone by negative regulation of kinetochore oscillation. Here the authors report that disrupting Kif18a function results in complete sterility in male but not in female mice. Histological examination reveals that Kif18a−/− testes exhibit severe developmental impairment of seminiferous tubules. Testis atrophy in Kif18a−/− mice is caused by perturbation of microtubule dynamics and spindle pole integrity, leading to chromosome congression defects during mitosis and meiosis. Depletion of KIF18A via RNAi causes mitotic arrest accompanied by unaligned chromosomes and increased microtubule nucleating centers in both GC-1 and HeLa cells. Prolonged depletion of KIF18A causes apoptosis due to perturbed microtubule dynamics. Further studies reveal that KIF18A silencing results in degradation of CENP-E and BubR1, which is accompanied by premature sister chromatid separation. KIF18A physically interacts with BubR1 and CENP-E, and this interaction is modulated during mitosis. Combined, the studies indicate that KIF18A is essential for normal chromosome congression during cell division and that the absence of KIF18A function causes severe defects in microtubule dynamics, spindle integrity, and checkpoint activation, leading to germinal cell aplasia in mice. PMID:20981276

New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment?

PubMed

Coqueret, Olivier

2003-02-01

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

PubMed

Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

PubMed Central

Dadke, Disha; Jarnik, Michael; Pugacheva, Elena N.; Singh, Mahendra K.; Golemis, Erica A.

2006-01-01

The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. PMID:16394104

Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

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

Ma, Yong-Cheng; Su, Nan; Shi, Xiao-Jing

2015-01-15

Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

PubMed Central

Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-01-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

PubMed Central

Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

2013-01-01

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

PubMed

Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

2013-09-03

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Induction of intracellular Ca2+ and pH changes in Sf9 insect cells by rhodojaponin-III, a natural botanic insecticide isolated from Rhododendron molle.

PubMed

Cheng, Xing-An; Xie, Jian-Jun; Hu, Mei-Ying; Zhang, Yan-Bo; Huang, Jing-Fei

2011-04-15

Many studies on intracellular calcium ([Ca2+](i)) and intracellular pH (pH(i)) have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+](i) and pH(i) and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+](i)) and intracellular pH (pH(i)) in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL) increase in [Ca2+](i) and pH(i) of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+](i), because completely treating Sf9 cells with CdCl(2) (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+](i) in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pH(i) showed similar changes with that of [Ca2+](i) on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+](i), cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

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.

G2 phase-specific proteins of HeLa cells.

PubMed Central

Al-Bader, A A; Orengo, A; Rao, P N

1978-01-01

The objective of this study was to determine if HeLa cells irreversibly arrested in G2 phase of the cell cycle by a brief exposure to a nitrosourea compound were deficient in certain proteins when compared with G2-synchronized cells. Total cellular proteins of G2-synchronized, G2-arrested, and S phase-synchronized cells were compared by two-dimensional polyacrylamide gel electrophoresis. The S phase cells differed from the G2-synchronized and G2-arrested cells by the absence of about 35 and 25 protein spots, respectively, of a total of nearly 150. At least nine protein spots in the molecular weight range of 4--5 X 10(4) that were present in the G2-synchronized cells were absent in both the G2-arrested and the S phase cells. Thus, these studies suggest that the missing proteins are probably necessary for the transition of cells from G2 phase to mitosis. Supplying the missing proteins to the G2-arrested cells by fusion with G2-synchronized cells facilitated the entry of the former into mitosis. Images PMID:282623

Control of G1 arrest after DNA damage.

PubMed Central

Kastan, M B; Kuerbitz, S J

1993-01-01

The temporal relationship between DNA damage and DNA replication may be critical in determining whether the genetic changes necessary for cellular transformation occur after DNA damage. Recent characterization of the mechanisms responsible for alterations in cell-cycle progression after DNA damage in our laboratory have implicated the p53 (tumor suppressor) protein in the G1 arrest that occurs after certain types of DNA damage. In particular, we found that levels of p53 protein increased rapidly and transiently after nonlethal doses of gamma irradiation (XRT) in hematopoietic cells with wild-type, but not mutant, p53 genes. These changes in p53 protein levels were temporally linked to a transient G1 arrest in these cells. Hematopoietic cells with mutant or absent p53 genes did not exhibit this G1 arrest, through they continued to demonstrate a G2 arrest. We recently extended these observations of a tight correlation between the status of the endogenous p53 genes and this G1 arrest after XRT and this cell-cycle alteration after XRT was then established by transfecting cells lacking endogenous p53 genes with a wild-type gene and observing acquisition of the G1 arrest and by transfecting cells processing endogenous wild-type p53 genes with a mutant p53 gene and observing loss of the G1 arrest after XRT. These observations and their significance for our understanding of the mechanisms of DNA damage-induced cellular transformation are discussed. PMID:8013425

Visualizing Vpr-Induced G2 Arrest and Apoptosis

PubMed Central

Murakami, Tomoyuki; Aida, Yoko

2014-01-01

Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the dynamics of the morphological changes that occur during Vpr-induced G2 arrest and apoptosis. PMID:24466265

Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

PubMed

Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

2013-08-01

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

Transcript and protein profiling identifies signaling, growth arrest, apoptosis, and NF-κB survival signatures following GNRH receptor activation

PubMed Central

Meyer, Colette; Sims, Andrew H; Morgan, Kevin; Harrison, Beth; Muir, Morwenna; Bai, Jianing; Faratian, Dana; Millar, Robert P; Langdon, Simon P

2013-01-01

GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60) in vitro and in vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene, CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60 in vitro, and p-NF-κB and IκBϵ were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation. PMID:23202794

T7 replisome directly overcomes DNA damage

NASA Astrophysics Data System (ADS)

Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

2015-12-01

Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

PubMed

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

PubMed

Robbins, Jonathan A; Absalon, Sabrina; Streva, Vincent A; Dvorin, Jeffrey D

2017-06-13

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G 1 -, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G 1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways. IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle. Copyright © 2017 Robbins et al.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

PubMed Central

Levasseur, Mark; Dumollard, Remi; Chambon, Jean-Philippe; Hebras, Celine; Sinclair, Maureen; Whitaker, Michael; McDougall, Alex

2013-01-01

The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/Ccdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates. PMID:24194472

A PAIR OF TRANSMEMBRANE RECEPTORS ESSENTIAL FOR THE RETENTION AND PIGMENTATION OF HAIR

PubMed Central

Han, Rong; Beppu, Hideyuki; Lee, Yun-Kyoung; Georgopoulos, Katia; Larue, Lionel; Li, En; Weiner, Lorin; Brissette, Janice L.

2012-01-01

Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division. PMID:22611050

Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

PubMed Central

Qiu, Hongming; Orr, F.William; Jensen, Derrek; Wang, Hui Helen; McIntosh, Alan R.; Hasinoff, Brian B.; Nance, Dwight M.; Pylypas, Susan; Qi, Ke; Song, Chun; Muschel, Ruth J.; Al-Mehdi, Abu-Bakr

2003-01-01

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by NG-nitro-l-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ∼55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. PMID:12547699

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.

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

PubMed

Hegde, Mahesh; Vartak, Supriya V; Kavitha, Chandagirikoppal V; Ananda, Hanumappa; Prasanna, Doddakunche S; Gopalakrishnan, Vidya; Choudhary, Bibha; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2017-05-31

Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost.

PubMed

Ding, Xiang; Zhu, Hongqing; Hou, Yiling; Hou, Wanru; Zhang, Nan; Fu, Lei

2017-01-01

The mechanism of the immunoregulatory activities of polysaccharide is still not clear. Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide.

A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA.

PubMed

Sozou, P D; Kirkwood, T B

2001-12-21

Human diploid fibroblast cells can divide for only a limited number of times in vitro, a phenomenon known as replicative senescence or the Hayflick limit. Variability in doubling potential is observed within a clone of cells, and between two sister cells arising from a single mitotic division. This strongly suggests that the process by which cells become senescent is intrinsically stochastic. Among the various biochemical mechanisms that have been proposed to explain replicative senescence, particular interest has been focussed on the role of telomere reduction. In the absence of telomerase--an enzyme switched off in normal diploid fibro-blasts-cells lose telomeric DNA at each cell division. According to the telomere hypothesis of cell senescence, cells eventually reach a critically short telomere length and cell cycle arrest follows. In support of this concept, forced expression of telomerase in normal fibroblasts appears to prevent cell senescence. Nevertheless, the telomere hypothesis in its basic form has some difficulty in explaining the marked stochastic variations seen in the replicative lifespans of individual cells within a culture, and there is strong empirical and theoretical support for the concept that other kinds of damage may contribute to cellular ageing. We describe a stochastic network model of cell senescence in which a primary role is played by telomere reduction but in which other mechanisms (oxidative stress linked particularly to mitochondrial damage, and nuclear somatic mutations) also contribute. The model gives simulation results that are in good agreement with published data on intra-clonal variability in cell doubling potential and permits an analysis of how the various elements of the stochastic network interact. Such integrative models may aid in developing new experimental approaches aimed at unravelling the intrinsic complexity of the mechanisms contributing to human cell ageing. Copyright 2001 Academic Press.

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

PubMed

Li, Huiyan; Peng, Xuan; Wang, Yating; Cao, Shirong; Xiong, Liping; Fan, Jinjin; Wang, Yihan; Zhuang, Shougang; Yu, Xueqing; Mao, Haiping

2016-09-01

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

Telomere Restriction Fragment (TRF) Analysis.

PubMed

Mender, Ilgen; Shay, Jerry W

2015-11-20

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al. , 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al. , 1990; Ouellette et al. , 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity.

PubMed

Brevini, T A L; Pennarossa, G; Vanelli, A; Maffei, S; Gandolfi, F

2012-03-01

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of contact time and force on monocyte adhesion to vascular endothelium.

PubMed Central

Rinker, K D; Prabhakar, V; Truskey, G A

2001-01-01

In this study we examined whether monocytic cell attachment to vascular endothelium was affected by elevating shear stress at a constant shear rate. Contact time, which is inversely related to the shear rate, was fixed and viscosity elevated with dextran to increase the shear stress (and hence the net force on the cell) independently of shear rate. At a fixed contact time, tethering frequencies increased, rolling velocities decreased, and median arrest durations increased with increasing shear stress. Rolling and short arrests (< 0.2 s) were well fit by a single exponential consistent with adhesion via the formation of a single additional bond. The cell dissociation constant, k(off), increased when the shear stress was elevated at constant shear rate. Firmly adherent cells arresting for at least 0.2 s were well fit by a stochastic model involving dissociation from multiple bonds. Therefore, at a fixed contact time and increasing shear stress, bonds formed more frequently for rolling cells resulting in more short arrests, and more bonds formed for firmly arresting cells resulting in longer arrest durations. Possible mechanisms for this increased adhesion include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. PMID:11259286

A Unifying Theory of Prostate Cancer

DTIC Science & Technology

2001-09-01

of cell cycle arrest or apoptosis, may lead to genomic instability and the development of cancer. 10 * One of our goals was to determine whether p53...and cell cycle arrest. Therefore, mechanisms responsible for upregulation and activation of p53 are intact in prostatic epithelial cells. We also...such as y-irradiation (Girinsky et al., 1995). The p53 protein is known to be a key regulator of cell cycle arrest and/or apoptosis (Wiman, 1997

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells.

PubMed

Park, Hyun Soo; Han, Min Ho; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hwang, Hye Jin; Park, Kun Young; Choi, Yung Hyun

2014-02-01

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Modulation of Medium pH by Caulobacter crescentus Facilitates Recovery from Uranium-Induced Growth Arrest

PubMed Central

Park, Dan M.

2014-01-01

The oxidized form of uranium [U(VI)] predominates in oxic environments and poses a major threat to ecosystems. Due to its ability to mineralize U(VI), the oligotroph Caulobacter crescentus is an attractive candidate for U(VI) bioremediation. However, the physiological basis for U(VI) tolerance is unclear. Here we demonstrated that U(VI) caused a temporary growth arrest in C. crescentus and three other bacterial species, although the duration of growth arrest was significantly shorter for C. crescentus. During the majority of the growth arrest period, cell morphology was unaltered and DNA replication initiation was inhibited. However, during the transition from growth arrest to exponential phase, cells with shorter stalks were observed, suggesting a decoupling between stalk development and the cell cycle. Upon recovery from growth arrest, C. crescentus proliferated with a growth rate comparable to that of a control without U(VI), although a fraction of these cells appeared filamentous with multiple replication start sites. Normal cell morphology was restored by the end of exponential phase. Cells did not accumulate U(VI) resistance mutations during the prolonged growth arrest, but rather, a reduction in U(VI) toxicity occurred concomitantly with an increase in medium pH. Together, these data suggest that C. crescentus recovers from U(VI)-induced growth arrest by reducing U(VI) toxicity through pH modulation. Our finding represents a unique U(VI) detoxification strategy and provides insight into how microbes cope with U(VI) under nongrowing conditions, a metabolic state that is prevalent in natural environments. PMID:25002429

Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

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

Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro, E-mail: fujii@sapmed.ac.j

2011-05-25

Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression ofmore » C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.« less

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage

PubMed Central

Manson, Margaret M.

2013-01-01

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G2/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G2/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G2/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53–/–, HCT116p21–/–, HT-29 and SW480). Flow cytometry confirmed that these lines underwent G2/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5–10 μM). In all eight lines, the majority of this arrest occurred at the G2/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G2/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

Palmitic acid-induced neuron cell cycle G2/M arrest and endoplasmic reticular stress through protein palmitoylation in SH-SY5Y human neuroblastoma cells.

PubMed

Hsiao, Yung-Hsuan; Lin, Ching-I; Liao, Hsiang; Chen, Yue-Hua; Lin, Shyh-Hsiang

2014-11-13

Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs) in the brain. An increase in SFAs, especially palmitic acid (PA), triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER) stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer's disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction.

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 prospective mesoderm genes twist, snail, and mef2 in penaeid shrimp.

PubMed

Wei, Jiankai; Glaves, Richard Samuel Elliot; Sellars, Melony J; Xiang, Jianhai; Hertzler, Philip L

2016-07-01

In penaeid shrimp, mesoderm forms from two sources: naupliar mesoderm founder cells, which invaginate during gastrulation, and posterior mesodermal stem cells called mesoteloblasts, which undergo characteristic teloblastic divisions. The primordial mesoteloblast descends from the ventral mesendoblast, which arrests in cell division at the 32-cell stage and ingresses with its sister dorsal mesendoblast prior to naupliar mesoderm invagination. The naupliar mesoderm forms the muscles of the naupliar appendages (first and second antennae and mandibles), while the mesoteloblasts form the mesoderm, including the muscles, of subsequently formed posterior segments. To better understand the mechanism of mesoderm and muscle formation in penaeid shrimp, twist, snail, and mef2 cDNAs were identified from transcriptomes of Penaeus vannamei, P. japonicus, P. chinensis, and P. monodon. A single Twist ortholog was found, with strong inferred amino acid conservation across all three species. Multiple Snail protein variants were detected, which clustered in a phylogenetic tree with other decapod crustacean Snail sequences. Two closely-related mef2 variants were found in P. vannamei. The developmental mRNA expression of these genes was studied by qPCR in P. vannamei embryos, larvae, and postlarvae. Expression of Pv-twist and Pv-snail began during the limb bud stage and continued through larval stages to the postlarva. Surprisingly, Pv-mef2 expression was found in all stages from the zygote to the postlarva, with the highest expression in the limb bud and protozoeal stages. The results add comparative data on the development of anterior and posterior mesoderm in malacostracan crustaceans, and should stimulate further studies on mesoderm and muscle development in penaeid shrimp.

Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells.

PubMed

Chou, Yu-Cheng; Chang, Meng-Ya; Wang, Mei-Jen; Liu, Hsin-Chung; Chang, Shu-Jen; Harnod, Tomor; Hung, Chih-Huang; Lee, Hsu-Tung; Shen, Chiung-Chyi; Chung, Jing-Gung

2017-01-01

Glioblastoma is the most common and aggressive primary brain malignancy. Phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, can induce apoptosis in many human cancer cells. Our previous study disclosed that PEITC induces apoptosis through the extrinsic pathway, dysfunction of mitochondria, reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress, and intrinsic (mitochondrial) pathway in human brain glioblastoma multiforme (GBM) 8401 cells. To the best of our knowledge, we first investigated the effects of PEITC on the genetic levels of GBM 8401 cells in vitro. PEITC may induce G0/G1 cell-cycle arrest through affecting the proteins such as cdk2, cyclin E, and p21 in GBM 8401 cells. Many genes associated with cell-cycle regulation of GBM 8401 cells were changed after PEITC treatment: 48 genes were upregulated and 118 were downregulated. The cell-division cycle protein 20 (CDC20), Budding uninhibited by benzimidazole 1 homolog beta (BUB1B), and cyclin B1 were downregulated, and clusterin was upregulated in GBM 8401 cells treated with PEITC. These changes of gene expression can provide the effects of PEITC on the genetic levels and potential biomarkers for glioblastoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 176-187, 2017. © 2015 Wiley Periodicals, Inc.

Differential Effects of Naja naja atra Venom on Immune Activity

PubMed Central

Kou, Jian-Qun; Han, Rong; Xu, Yin-Li; Ding, Xiao-Lan; Wang, Shu-Zhi; Chen, Cao-Xin; Ji, Hong-Zhang; Ding, Zhi-Hui; Qin, Zheng-Hong

2014-01-01

Previous studies reported that Naja naja atra venom (NNAV) inhibited inflammation and adjuvant arthritis. Here we investigated the role of NNAV in regulation of immune responses in mice. Oral administration of NNAV to normal mice showed significant increase in natural killer cell activity, B lymphocyte proliferation stimulated by lipopolysaccharides, and antibody production in response to sheep red blood cells. Meanwhile, NNAV markedly decreased T lymphocyte proliferation stimulated by concanavalin A, arrested the cell cycle at G0/G1 phase, and suppressed CD4 and CD8 T cell divisions. Furthermore, NNAV inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reaction. This modulation of immune responses may be partly attributed to the selective increase in Th1 and Th2 cytokines (IFN-γ, IL-4) secretion and inhibition of Th17 cytokine (IL-17) production. In dexamethasone-induced immunosuppressed mice, NNAV restored the concentration of serum IgG and IgM, while decreasing the percentage of CD4 and CD8 T-cell subsets. These results indicate that NNAV enhances the innate and humoral immune responses while inhibiting CD4 Th17 and CD8 T cell actions, suggesting that NNAV could be a potential therapeutic agent for autoimmune diseases. PMID:25024726

Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

PubMed

Di Genova, Bruno M; da Silva, Richard C; da Cunha, Júlia P C; Gargantini, Pablo R; Mortara, Renato A; Tonelli, Renata R

2017-07-01

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Dietary Restriction and Fasting Arrest B and T Cell Development and Increase Mature B and T Cell Numbers in Bone Marrow

PubMed Central

Shushimita, Shushimita; de Bruijn, Marjolein J. W.; de Bruin, Ron W. F.; IJzermans, Jan N. M.; Hendriks, Rudi W.; Dor, Frank J. M. F.

2014-01-01

Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many “neuronal and surgery related damaging phenomena” such as Parkinson’s disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined. PMID:24504160

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

PubMed

Weinert, T A; Hartwell, L H

1990-12-01

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

Analysis of Giant-nucleated Cell Formation Following X-ray and Proton Irradiations

NASA Astrophysics Data System (ADS)

Almahwasi, Ashraf Abdu

Radiation-induced genetic instability has been observed in survivors of irradiated cancerous and normal cells in vitro and in vivo and has been determined in different forms, such as delayed cell death, chromosomal aberration or mutation. A well defined and characterized normal human-diploid AG1522 fibroblast cell line was used to study giant-nucleated cell (GCs) formation as the ultimate endpoint of this research. The average nuclear cross-sectional areas of the AG1522 cells were measured in mum2. The doubling time required by the AG1522 cells to divide was measured. The potential toxicity of the Hoechst dye at a working concentration on the live AG1522 cells was assessed. The yield of giant cells was determined at 7, 14 and 21 days after exposure to equivalent clinical doses of 0.2, 1 or 2 Gy of X-ray or proton irradiation. Significant differences were found to exist between X-ray or proton irradiation when compared with sham-irradiated control populations. The frequency of GCs induced by X-rays was also compared to those formed in proton irradiated cultures. The results confirm that 1 Gy X-rays are shown to induce higher rates of mitotically arrested GCs, increasing continually over time up to 21 days post-irradiation. The yield of GCs was significantly greater (10%) compared to those formed in proton populations (2%) 21 days postirradiation. The GCs can undergo a prolonged mitotic arrest that significantly increases the length of cell cycle. The arrest of GCs at the mitotic phase for longer periods of time might be indicative of a strategy for cell survival, as it increases the time available for DNA repair and enables an alternative route to division for the cells. However, the reduction in their formation 21 days after both types of radiation might favour GCs formation, ultimately contributing to carcinogenesis or cancer therapy resistance. The X-ray experiments revealed a dose-dependent increase in the GCs up to 14 days after irradiation. Although the proton irradiation was less efficient in producing GCs, their frequency was elevated in a dose-dependent manner 7 days after irradiation, with persistent expression of nuclear deformity as an indicator of genetic instability. In addition to the quantification of the GCs, the proliferation of a small fraction of giant cells formed at 14 days after 0.2 Gy of proton irradiation was observed to divide into asymmetrical, normal-sized daughter cells. These results might have important implications in evaluating risk estimates, or could act as a potential radioprotective assay for a dose-limiting parameter for delayed effects in healthy tissues during radiation therapy treatment.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

PubMed Central

Waraky, Ahmed; Akopyan, Karen; Parrow, Vendela; Strömberg, Thomas; Axelson, Magnus; Abrahmsén, Lars; Lindqvist, Arne; Larsson, Olle; Aleem, Eiman

2014-01-01

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP. PMID:25268741

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

PubMed

Tsai, Tsung-Chang; Huang, Hui-Pei; Chang, Kai-Ting; Wang, Chau-Jong; Chang, Yun-Ching

2017-04-01

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL -1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017. © 2016 Wiley Periodicals, Inc.

Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

PubMed Central

Yumoto, Takahito; Nakadate, Kazuhiko; Nakamura, Yuki; Sugitani, Yoshinobu; Sugitani-Yoshida, Reiko; Ueda, Shuichi; Sakakibara, Shin-ichi

2013-01-01

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs. PMID:24260314

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

PubMed

Liu, Chang Ching; Ma, Dong Liang; Yan, Ting-Dong; Fan, XiuBo; Poon, Zhiyong; Poon, Lai-Fong; Goh, Su-Ann; Rozen, Steve G; Hwang, William Ying Khee; Tergaonkar, Vinay; Tan, Patrick; Ghosh, Sujoy; Virshup, David M; Goh, Eyleen L K; Li, Shang

2016-10-01

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484. © 2016 AlphaMed Press.

Src Homology 2-Domain Containing Leukocyte-Specific Phosphoprotein of 76 kDa (SLP-76) Is Required for Optimal CXCR4-Stimulated T Lymphocyte Firm Arrest to ICAM-1 Under Shear Flow

PubMed Central

Lee, Dooyoung; Kim, Jiyeon; Baker, Rebecca G.; Koretzky, Gary A.; Hammer, Daniel A.

2014-01-01

Summary Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adaptor protein SLP-76 in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α SDF-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in this T cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking. PMID:22806433

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation*

PubMed Central

Geng, Feng; Tang, Li; Li, Yun; Yang, Lu; Choi, Kyoung-Soo; Kazim, A. Latif; Zhang, Yuesheng

2011-01-01

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and β-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet. PMID:21778226

Lentiviral Delivery of HIV-1 Vpr Protein Induces Apoptosis in Transformed Cells

NASA Astrophysics Data System (ADS)

Stewart, Sheila A.; Poon, Betty; Jowett, Jeremy B. M.; Xie, Yiming; Chen, Irvin S. Y.

1999-10-01

Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G2/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.

Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection

PubMed Central

Hayashi, Makoto T.; Cesare, Anthony J.; Rivera, Teresa; Karlseder, Jan

2015-01-01

Tumour formation is blocked by two barriers, replicative senescence and crisis1. Senescence is triggered by short telomeres and is bypassed by disruption of tumour suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbor unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remained elusive. We show that cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. The phenotype was induced by loss of p53 function, and suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating such fusions as the underlying cause. Exacerbation of mitotic telomere deprotection by partial TRF2 knockdown2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population. PMID:26108857

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

NASA Technical Reports Server (NTRS)

Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

2001-01-01

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost

PubMed Central

Ding, Xiang; Zhu, Hongqing; Hou, Yiling; Hou, Wanru; Zhang, Nan; Fu, Lei

2017-01-01

Background: The mechanism of the immunoregulatory activities of polysaccharide is still not clear. Materials and Methods: Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. Results: These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. Conclusion: An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. SUMMARY Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide. PMID:28839373

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

PubMed Central

Morino, Masayuki; Nukina, Kohei; Sakaguchi, Hiroki; Maeda, Takeshi; Takahara, Michiyo; Shiomi, Yasushi; Nishitani, Hideo

2015-01-01

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis. PMID:25798850

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

PubMed Central

Kriegs, Malte; Kasten-Pisula, Ulla; Riepen, Britta; Hoffer, Konstantin; Struve, Nina; Myllynen, Laura; Braig, Friederike; Binder, Mascha; Rieckmann, Thorsten; Grénman, Reidar; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai

2016-01-01

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells. PMID:27281611

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

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

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry ofmore » γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.« less

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest.

PubMed

Pérez-Sánchez, Gilberto; Jiménez, Adriana; Quezada-Ramírez, Marco A; Estudillo, Enrique; Ayala-Sarmiento, Alberto E; Mendoza-Hernández, Guillermo; Hernández-Soto, Justino; Hernández-Hernández, Fidel C; Cázares-Raga, Febe E; Segovia, Jose

2018-05-01

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B), and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional the level in proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability. © 2017 Wiley Periodicals, Inc.

Cell cycle perturbation induced by gemcitabine in human tumor cells in cell culture, xenografts and bladder cancer patients: implications for clinical trial designs combining gemcitabine with a Chk1 inhibitor.

PubMed

Montano, Ryan; Khan, Nadeem; Hou, Huagang; Seigne, John; Ernstoff, Marc S; Lewis, Lionel D; Eastman, Alan

2017-09-15

Gemcitabine irreversibly inhibits ribonucleotide reductase and induces S phase arrest but whether this occurs in tumors in mice or patients has not been established. Tumor cells in culture were incubated with gemcitabine for 6 h to approximate the administration schedule in a patient. Concentrations that induced persistent S phase arrest thereafter correlated with cell killing. Administration of gemcitabine to mice also demonstrated a persistent S phase arrest in their tumor. The minimum dose that induced almost complete S phase arrest after 24 h (40 mg/kg) was well below the maximum tolerated dose in mice. S phase arrest was also observed in tumors of bladder cancer patients receiving gemcitabine. The Chk1 inhibitor MK-8776 sensitized cells to gemcitabine with the greatest cell killing when added 18 h after gemcitabine. In mice, the administration of MK-8776 18 h after gemcitabine elicited positivity for the DNA damage marker γH2AX; this also occurred at relatively low dose (40 mg/kg) gemcitabine. Hence, in both cell culture and xenografts, MK-8776 can markedly enhance cell killing of cells reversibly arrested in S phase by gemcitabine. Some cell lines are hypersensitive to MK-8776 as monotherapy, but this was not observed in xenograft models. Effective monotherapy requires a higher dose of Chk1 inhibitor, and target inhibition over a longer time period as compared to its use in combination. These results have important implications for combining Chk1 inhibitors with gemcitabine and suggest that Chk1 inhibitors with increased bioavailability may have improved efficacy both in combination and as monotherapy.

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway.

PubMed

Xu, Yong-Jie; Singh, Amanpreet; Alter, Gerald M

2016-11-01

Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood. While screening for new fission yeast mutants that are sensitive to replication stress, we identified a novel mutation in the erg11 gene encoding the enzyme sterol-14α-demethylase in the ergosterol biosynthesis pathway that dramatically sensitizes the cells to chronic HU treatment. Surprisingly, HU mainly arrests the erg11 mutant cells in cytokinesis, not in S phase. Unlike the reversible S phase arrest in wild-type cells, the cytokinesis arrest induced by HU is relatively stable and occurs at low doses of the drug, which likely explains the remarkable sensitivity of the mutant to HU. We also show that the mutation causes sterol deficiency, which may predispose the cells to the cytokinesis arrest and lead to cell death. We hypothesize that in addition to the RNR, HU may have a secondary unknown target(s) inside cells. Identification of such a target(s) may greatly improve the chemotherapies that employ HU or help to expand the clinical usage of this drug for additional pathological conditions. Copyright © 2016 by the Genetics Society of America.

Developmentally arrested structures preceding cerebellar tumors in von Hippel–Lindau disease

PubMed Central

Shively, Sharon B; Falke, Eric A; Li, Jie; Tran, Maxine G B; Thompson, Eli R; Maxwell, Patrick H; Roessler, Erich; Oldfield, Edward H; Lonser, Russell R; Vortmeyer, Alexander O

2011-01-01

There is increasing evidence that suggests that knockout of tumor-suppressor gene function causes developmental arrest and protraction of cellular differentiation. In the peripheral nervous system of patients with the tumor-suppressor gene disorder, von Hippel–Lindau disease, we have demonstrated developmentally arrested structural elements composed of hemangioblast progenitor cells. Some developmentally arrested structural elements progress to a frank tumor, hemangioblastoma. However, in von Hippel–Lindau disease, hemangioblastomas are frequently observed in the cerebellum, suggesting an origin in the central nervous system. We performed a structural and topographic analysis of cerebellar tissues obtained from von Hippel–Lindau disease patients to identify and characterize developmentally arrested structural elements in the central nervous system. We examined the entire cerebella of five tumor-free von Hippel–Lindau disease patients and of three non-von Hippel–Lindau disease controls. In all, 9 cerebellar developmentally arrested structural elements were detected and topographically mapped in 385 blocks of von Hippel–Lindau disease cerebella. No developmentally arrested structural elements were seen in 214 blocks from control cerebella. Developmentally arrested structural elements are composed of poorly differentiated cells that express hypoxia-inducible factor (HIF)2α, but not HIF1α or brachyury, and preferentially involve the molecular layer of the dorsum cerebelli. For the first time, we identify and characterize developmentally arrested structural elements in the central nervous system of von Hippel–Lindau patients. We provide evidence that developmentally arrested structural elements in the cerebellum are composed of developmentally arrested hemangioblast progenitor cells in the molecular layer of the dorsum cerebelli. PMID:21499240

Valproic acid exhibits different cell growth arrest effect in three HPV-positive/negative cervical cancer cells and possibly via inducing Notch1 cleavage and E6 downregulation.

PubMed

Feng, Shuyu; Yang, Yue; Lv, Jingyi; Sun, Lichun; Liu, Mingqiu

2016-07-01

We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.

SLP-76 is required for optimal CXCR4-stimulated T lymphocyte firm arrest to ICAM-1 under shear flow.

PubMed

Lee, Dooyoung; Kim, Jiyeon; Baker, Rebecca G; Koretzky, Gary A; Hammer, Daniel A

2012-10-01

Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T-cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adapter protein SLP-76 (Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa) in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T-cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α (stromal cell-derived factor-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in T-cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

PubMed

Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

2006-03-01

Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background

PubMed Central

Cook, Matthew S.; Munger, Steven C.; Nadeau, Joseph H.; Capel, Blanche

2011-01-01

Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1Ter/Ter mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1Ter/Ter mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1Ter/Ter; Bax–/– double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1Ter/Ter germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27Kip1 and p21Cip1. P27Kip1 and P21Cip1 protein are both significantly decreased in Dnd1Ter/Ter germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG, SOX2 and OCT4. Consistent with their ability to rescue cell cycle arrest, C57BL/6J germ cells overexpress negative regulators of the cell cycle relative to 129/SvJ. This work suggests that reprogramming of pluripotency in germ cells and prevention of tumor formation requires cell cycle arrest, and that differences in the balance of cell cycle regulators between 129/SvJ and C57BL/6 might underlie differences in tumor susceptibility. PMID:21115610

Hemodynamic Forces Tune the Arrest, Adhesion, and Extravasation of Circulating Tumor Cells.

PubMed

Follain, Gautier; Osmani, Naël; Azevedo, Ana Sofia; Allio, Guillaume; Mercier, Luc; Karreman, Matthia A; Solecki, Gergely; Garcia Leòn, Marìa Jesùs; Lefebvre, Olivier; Fekonja, Nina; Hille, Claudia; Chabannes, Vincent; Dollé, Guillaume; Metivet, Thibaut; Hovsepian, François Der; Prudhomme, Christophe; Pichot, Angélique; Paul, Nicodème; Carapito, Raphaël; Bahram, Siamak; Ruthensteiner, Bernhard; Kemmling, André; Siemonsen, Susanne; Schneider, Tanja; Fiehler, Jens; Glatzel, Markus; Winkler, Frank; Schwab, Yannick; Pantel, Klaus; Harlepp, Sébastien; Goetz, Jacky G

2018-04-09

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth. Copyright © 2018 Elsevier Inc. All rights reserved.

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

PubMed

Yin, Jiuheng; Sheng, Baifa; Han, Bin; Pu, Aimin; Yang, Kunqiu; Li, Ping; Wang, Qimeng; Xiao, Weidong; Yang, Hua

2016-05-01

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation. © 2016 International Federation for Cell Biology.

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

NASA Technical Reports Server (NTRS)

Enebo, D. J.; Fattaey, H. K.; Moos, P. J.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1994-01-01

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition.

Traditional Chinese medicine herbal mixture LQ arrests FUCCI-expressing HeLa cells in G₀/G₁ phase in 2D plastic, 2.5D Matrigel, and 3D Gelfoam culture visualized with FUCCI imaging.

PubMed

Zhang, Lei; Wu, Chengyu; Bouvet, Michael; Yano, Shuya; Hoffman, Robert M

2015-03-10

We used the fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after treatment of FUCCI-expressing HeLa cells (FUCCI-HeLa) with a traditional Chinese medicine (TCM) herbal mixture LQ, previously shown to have anti-tumor and anti-metastatic activity in mouse models. Paclitaxel was used as the positive control. In 2D monolayer culture, the untreated control had approximately 45% of the cells in S/G₂/M phase. In contrast, the LQ-treated cells (9 mg/ml) were mostly in the G₀/G₁ (>90%) after 72 hours. After treatment with paclitaxel (0.01 μm), for 72 hours, 95% of the cells were in S/G₂/M. In 2.5D Matrigel culture, the colonies in the untreated control group had 40% of the cells in S/G₂/M. LQ arrested the cells in G₀/G₁ after 72 hours. Paclitaxel arrested almost all the cells in S/G₂/M after 72 hours. In 3D Gelfoam culture, the untreated control culture had approximately 45% of cells in G₂/M. In contrast, the LQ-treated cells were mostly in G₀/G₁ phase (>80%) after 72 hours treatment. Paclitaxel resulted in 90% of the cells arrested in S/G₂/M after 72 hours. The present report suggests the non-toxic LQ has potential to maintain cancers in a quiescent state for long periods of time.

Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide induced genotoxicity in human lymphocyte culture.

PubMed

Paul, Rajkumar; Kulkarni, Paresh; Ganesh, Narayan

2011-01-01

Diets rich in fruits and vegetables have been associated with reduced risks for many types of cancers. Avocado (Persea americana Mill.) is a widely consumed fruit containing many cancer preventing nutrients, vitamins and phytochemicals. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with 50% Methanol from avocado fruits help in proliferation of human lymphocyte cells and decrease chromosomal aberrations induced by cyclophosphamide. Among three concentrations (100 mg, 150 mg and 200 mg per Kg Body Weight), the most effective conc. of extract was 200 mg/Kg Body Wt. It decreased significant level of numerical and structural aberrations (breaks, premature centromeric division etc. up to 88%, p < 0.0001)), and accrocentric associtation within D & G group (up to 78%, p = 0.0008). These studies suggest that phytochemicals from the avocado fruit can be utilized for making active chemoprotective ingredient for lowering the side effect of chemotherapy like cyclophosphamide in cancer therapy.

Multi-functional roles of a soldier-specific volatile as a worker arrestant, primer pheromone and an antimicrobial agent in a termite.

PubMed

Mitaka, Yuki; Mori, Naoki; Matsuura, Kenji

2017-07-26

Division of labour in eusocial insects is characterized by efficient communication systems based on pheromones. Among such insects, termites have evolved specialized sterile defenders, called soldiers. Because they are incapable of feeding themselves, it has been suggested that soldiers are sustained by workers and emit the pheromone arresting workers. However, such a soldier pheromone has not been identified in any termite species, and the details of the soldier-worker interaction remain to be explored. Here, we identified a soldier-specific volatile sesquiterpene as a worker arrestant, which also acts as a primer pheromone regulating soldier differentiation and fungistatic agent in a termite Reticulitermes speratus Chemical analyses revealed that (-)- β -elemene is the major component of soldier extract, and its authentic standard exhibited arrestant activity to workers and inhibited the differentiation from workers to soldiers. This compound also showed fungistatic activity against entomopathogenic fungi. These suggest that (-)- β -elemene secreted by soldiers acts not only as a worker arrestant but also as one component of inhibitory primer pheromone and an anti-pathogenic agent. Our study provides novel evidence supporting the multi-functionality of termite soldier pheromone and provides new insights into the role of soldiers and the evolutionary mechanisms of pheromone compounds. © 2017 The Author(s).

Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase.

PubMed

Sun, Pei; Wu, Haoyang; Huang, Jiali; Xu, Ying; Yang, Feng; Zhang, Qi; Xu, Xingang

2018-05-22

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the swine industry. p53 protein exists in a wide variety of animal cells, which is involved in cell cycle regulation, apoptosis, cell differentiation and other biological functions. In this study, we investigated the effects of PEDV infection on the cell cycle of Vero cells and p53 activation. The results demonstrated that PEDV infection induces cell cycle arrest at G0/G1 phase in Vero cells, while UV-inactivated PEDV does not cause cell cycle arrest. PEDV infection up-regulates the levels of p21, cdc2, cdk2, cdk4, Cyclin A protein and down-regulates Cyclin E protein. Further research results showed that inhibition of p53 signaling pathway can reverse the cell cycle arrest in G0/G1 phase induced by PEDV infection and cancel out the up-regulation of p21 and corresponding Cyclin/cdk mentioned above. In addition, PEDV infection of the cells synchronized in various stages of cell cycle showed that viral subgenomic RNA and virus titer were higher in the cells released from G0/G1 phase synchronized cells than that in the cells released from the G1/S phase and G2/M phase synchronized or asynchronous cells after 18 h p.i.. This is the first report to demonstrate that the p53-dependent pathway plays an important role in PEDV induced cell cycle arrest and beneficially contributes to viral infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Antagonizing functions of BARD1 and its alternatively spliced variant BARD1δ in telomere stability.

PubMed

Pilyugin, Maxim; André, Pierre-Alain; Ratajska, Magdalena; Kuzniacka, Alina; Limon, Janusz; Tournier, Benjamin B; Colas, Julien; Laurent, Geoff; Irminger-Finger, Irmgard

2017-02-07

Previous reports have shown that expression of BARD1δ, a deletion-bearing isoform of BARD1, correlates with tumor aggressiveness and progression. We show that expression of BARD1δ induces cell cycle arrest in vitro and in vivo in non-malignant cells. We investigated the mechanism that leads to proliferation arrest and found that BARD1δ overexpression induced mitotic arrest with chromosome and telomere aberrations in cell cultures, in transgenic mice, and in cells from human breast and ovarian cancer patients with BARD1 mutations. BARD1δ binds more efficiently than BARD1 to telomere binding proteins and causes their depletion from telomeres, leading to telomere and chromosomal instability. While this induces cell cycle arrest, cancer cells lacking G2/M checkpoint controls might continue to proliferate despite the BARD1δ-induced chromosomal instability. These features of BARD1δ may make it a genome permutator and a driver of continuous uncontrolled proliferation of cancer cells.

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

PubMed Central

Rai, Urvashi; Najm, Fadi

2017-01-01

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle. PMID:28339487

Molecular Analysis of the Inheritance of Transcriptional Silencing

DTIC Science & Technology

2007-04-01

arrest, to synchronize the cells for hydroxyurea (HU) addition, and then released into an alpha factor/0.2M HU arrest (Fig. 1A). 2 hours after release...restrictive conditions to degrade both Sir1td and Asf1td proteins. Cells were then release for 4 hours into 0.2M hydroxyurea (HU), an early S phase arrest...on three experiments. The “Block” row describes the cell cycle inhibitor used in each time point (alpha factor, hydroxyurea and nocodozole). 24

Development of the follicle complex and oocyte staging in red drum, Sciaenops ocellatus Linnaeus, 1776 (Perciformes, Sciaenidae).

PubMed

Grier, Harry J

2012-08-01

Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One-Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full-Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II). The last stage, Ovulation, is the emergence of the oocyte from the follicle when it becomes an egg or ovum. Copyright © 2012 Wiley Periodicals, Inc.

G1 arrest and differentiation can occur independently of Rb family function

PubMed Central

Wirt, Stacey E.; Adler, Adam S.; Gebala, Véronique; Weimann, James M.; Schaffer, Bethany E.; Saddic, Louis A.; Viatour, Patrick; Vogel, Hannes; Chang, Howard Y.; Meissner, Alex

2010-01-01

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9–11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway. PMID:21059851

Mechanical entrapment is insufficient and intercellular adhesion is essential for metastatic cell arrest in distant organs.

PubMed

Glinskii, Olga V; Huxley, Virginia H; Glinsky, Gennadi V; Pienta, Kenneth J; Raz, Avraham; Glinsky, Vladislav V

2005-05-01

In this report, we challenge a common perception that tumor embolism is a size-limited event of mechanical arrest, occurring in the first capillary bed encountered by blood-borne metastatic cells. We tested the hypothesis that mechanical entrapment alone, in the absence of tumor cell adhesion to blood vessel walls, is not sufficient for metastatic cell arrest in target organ microvasculature. The in vivo metastatic deposit formation assay was used to assess the number and location of fluorescently labeled tumor cells lodged in selected organs and tissues following intravenous inoculation. We report that a significant fraction of breast and prostate cancer cells escapes arrest in a lung capillary bed and lodges successfully in other organs and tissues. Monoclonal antibodies and carbohydrate-based compounds (anti-Thomsen-Friedenreich antigen antibody, anti-galectin-3 antibody, modified citrus pectin, and lactulosyl-l-leucine), targeting specifically beta-galactoside-mediated tumor-endothelial cell adhesive interactions, inhibited by >90% the in vivo formation of breast and prostate carcinoma metastatic deposits in mouse lung and bones. Our results indicate that metastatic cell arrest in target organ microvessels is not a consequence of mechanical trapping, but is supported predominantly by intercellular adhesive interactions mediated by cancer-associated Thomsen-Friedenreich glycoantigen and beta-galactoside-binding lectin galectin-3. Efficient blocking of beta-galactoside-mediated adhesion precludes malignant cell lodging in target organs.

Invasion of Peripheral Immune Cells into Brain Parenchyma after Cardiac Arrest and Resuscitation.

PubMed

Zhang, Can; Brandon, Nicole R; Koper, Kerryann; Tang, Pei; Xu, Yan; Dou, Huanyu

2018-06-01

Although a direct link has long been suspected between systemic immune responses and neuronal injuries after stroke, it is unclear which immune cells play an important role. A question remains as to whether the blood brain barrier (BBB) is transiently disrupted after circulatory arrest to allow peripheral immune cells to enter brain parenchyma. Here, we developed a clinically relevant cardiac arrest and resuscitation model in mice to investigate the BBB integrity using noninvasive magnetic resonance imaging. Changes in immune signals in the brain and periphery were assayed by immunohistochemistry and flow cytometry. Quantitative variance maps from T1-weighted difference images before and after blood-pool contrast clearance revealed BBB disruptions immediately after resuscitation and one day after reperfusion. Time profiles of hippocampal CA1 neuronal injuries correlated with the morphological changes of microglia activation. Cytotoxic T cells, CD11b + CD11c + dendritic cells, and CD11b + CD45 +hi monocytes and macrophages were significantly increased in the brain three days after cardiac arrest and resuscitation, suggesting direct infiltration of these cells following the BBB disruption. Importantly, these immune cell changes were coupled with a parallel increase in the same subset of immune cell populations in the bone marrow and blood. We conclude that neurovascular breakdown during the initial reperfusion phase contributes to the systemic immune cell invasion and subsequent neuropathogenesis affecting the long-term outcome after cardiac arrest and resuscitation.

The long non-coding RNA GAS5 differentially regulates cell cycle arrest and apoptosis through activation of BRCA1 and p53 in human neuroblastoma

PubMed Central

Mazar, Joseph; Rosado, Amy; Shelley, John; Marchica, John; Westmoreland, Tamarah J

2017-01-01

The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death. PMID:28035057

Exposure cell number during feeder cell growth-arrest by Mitomycin C is a critical pharmacological aspect in stem cell culture system.

PubMed

Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

2016-01-01

Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10μg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays. Copyright © 2016 Elsevier Inc. All rights reserved.

A THERMODYNAMIC ANALYSIS OF MITOTIC SPINDLE EQUILIBRIUM AT ACTIVE METAPHASE

PubMed Central

Stephens, R. E.

1973-01-01

The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoué. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55–65 kcal/mol and +197–233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoué and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoué, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D2O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D2O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoué's earlier contention that D2O treatment can modify the available spindle pool. PMID:4734864

Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.

PubMed

Monson, Rita E; Tashiro, Yosuke; Salmond, George P C

2016-09-01

Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.

6-Mercaptopurine (6-MP) induces cell cycle arrest and apoptosis of neural progenitor cells in the developing fetal rat brain.

PubMed

Kanemitsu, H; Yamauchi, H; Komatsu, M; Yamamoto, S; Okazaki, S; Uchida, K; Nakayama, H

2009-01-01

6-Mercaptopurine (6-MP), an analogue of hypoxanthine, is used in the therapy of acute lymphoblastic leukemia and causes fetal neurotoxicity. To clarify the mechanisms of 6-MP-induced fetal neurotoxicity leading to the cell cycle arrest and apoptosis of neural progenitor cells, pregnant rats were treated with 50 mg/kg 6-MP on embryonic day (E) 13, and the fetal telencephalons were examined at 12 to 72 h (h) after treatment. Flow-cytometric analysis confirmed an accumulation of cells at G2/M, S, and sub-G1 (apoptotic cells) phases from 24 to 72 h. The number of phosphorylated histone H3-positive cells (mitotic cells) decreased from 36 to 72 h, and the phosphorylated (active) form of p53 protein, which is a mediator of apoptosis and cell cycle arrest, increased from 24 to 48 h. An executor of p53-mediated cell cycle arrest, p21, showed intense overexpression at both the mRNA and protein levels from 24 to 72 h. Cdc25A protein, which is needed for the progression of S phase, decreased at 36 and 48 h. In addition, phosphorylated cdc2 protein, which is an inactive form of cdc2 necessary for G2/M progression, increased from 24 to 48 h. These results suggest that 6-MP induced G2/M arrest, delayed S-phase progression, and finally induced apoptosis of neural progenitor cells mediated by p53 in the fetal rat telencephalon.

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.

Characterization of a Putative Spindle Assembly Checkpoint Kinase Mps1, Suggests Its Involvement in Cell Division, Morphogenesis and Oxidative Stress Tolerance in Candida albicans

PubMed Central

Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus. PMID:25025778

Characterization of a putative spindle assembly checkpoint kinase Mps1, suggests its involvement in cell division, morphogenesis and oxidative stress tolerance in Candida albicans.

PubMed

Kamthan, Mohan; Nalla, Vijaya Kumar; Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1 mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1 and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.

Suppression of Akt-mediated HDAC3 expression and CDK2 T39 phosphorylation by a bichalcone analog contributes to S phase retardation of cancer cells.

PubMed

Hung, Kuang-Chen; Lin, Meng-Liang; Hsu, Shih-Wei; Lee, Chuan-Chun; Huang, Ren-Yu; Wu, Tian-Shung; Chen, Shih-Shun

2018-06-15

Targeting cell cycle regulators has been a suggested mechanism for therapeutic cancer strategies. We report here that the bichalcone analog TSWU-CD4 induces S phase arrest of human cancer cells by inhibiting the formation of cyclin A-phospho (p)-cyclin-dependent kinase 2 (CDK2, threonine [Thr] 39) complexes, independent of mutant p53 expression. Ectopic expression of CDK2 (T39E), which mimics phosphorylation of the Thr 39 residue of CDK2, partially rescues the cells from TSWU-CD4-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TSWU-CD4-triggered S phase arrest. Decreased histone deacetylase 3 (HDAC3) expression after TSWU-CD4 treatment was demonstrated, and TSWU-CD4 induced S phase arrest and inhibitory effects on cyclin A expression and CDK2 Thr 39 phosphorylation, while cyclin A-p-CDK2 (Thr 39) complex formation was suppressed by ectopic wild-type HDAC3 expression. The co-transfection of CDK2 (T39E) along with HDAC3 completely restored cyclin A expression, Thr 39-phosphorylated CDK2, cyclin A-p-CDK2 (Thr 39) complex formation, and the S phase population to normal levels. Protein kinase B (Akt) inactivation was required for TSWU-CD4-induced S phase cell cycle arrest, because constitutively active Akt1 blocks the induction of S phase arrest and the suppression of cyclin A and HDAC3 expression, CDK2 Thr 39 phosphorylation, and cyclin A-p-CDK2 (Thr 39) complex formation by TSWU-CD4. Taken together, our results indicate that TSWU-CD4 induces S phase arrest by inhibiting Akt-mediated HDAC3 expression and CDK2 Thr 39 phosphorylation to suppress the formation of cyclin A-p-CDK2 (Thr 39) complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

PubMed

Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation

PubMed Central

Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania

2016-01-01

The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc−/−) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc−/− newborns but rescued in G3 Terc−/−/p21−/− mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

PubMed

The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

2015-01-06

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

PubMed

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-03-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism ( mog ) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf ( rl ) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD ( PPD ) that regulates arrest of cell divisions in meristematic cells . We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

cdc-25.2, a C. elegans ortholog of cdc25, is required to promote oocyte maturation.

PubMed

Kim, Jiyoung; Kawasaki, Ichiro; Shim, Yhong-Hee

2010-03-15

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

Characterization of KIF11 as a novel prognostic biomarker and therapeutic target for oral cancer.

PubMed

Daigo, Kayo; Takano, Atsushi; Thang, Phung Manh; Yoshitake, Yoshihiro; Shinohara, Masanori; Tohnai, Iwau; Murakami, Yoshinori; Maegawa, Jiro; Daigo, Yataro

2018-01-01

Oral cancer has a high mortality rate, and its incidence is increasing gradually worldwide. As the effectiveness of standard treatments is still limited, the development of new therapeutic strategies is eagerly awaited. Kinesin family member 11 (KIF11) is a motor protein required for establishing a bipolar spindle in cell division. The role of KIF11 in oral cancer is unclear. Therefore, the present study aimed to assess the role of KIF11 in oral cancer and evaluate its role as a prognostic biomarker and therapeutic target for treating oral cancer. Immunohistochemical analysis demonstrated that KIF11 was expressed in 64 of 99 (64.6%) oral cancer tissues but not in healthy oral epithelia. Strong KIF11 expression was significantly associated with poor prognosis among oral cancer patients (P=0.034), and multivariate analysis confirmed its independent prognostic value. In addition, inhibition of KIF11 expression by transfection of siRNAs into oral cancer cells or treatment of cells with a KIF11 inhibitor significantly suppressed cell proliferation, probably through G2/M arrest and subsequent induction of apoptosis. These results suggest that KIF11 could be a potential prognostic biomarker and therapeutic target for oral cancer.

Characterization of KIF11 as a novel prognostic biomarker and therapeutic target for oral cancer

PubMed Central

Daigo, Kayo; Takano, Atsushi; Thang, Phung Manh; Yoshitake, Yoshihiro; Shinohara, Masanori; Tohnai, Iwau; Murakami, Yoshinori; Maegawa, Jiro; Daigo, Yataro

2018-01-01

Oral cancer has a high mortality rate, and its incidence is increasing gradually worldwide. As the effectiveness of standard treatments is still limited, the development of new therapeutic strategies is eagerly awaited. Kinesin family member 11 (KIF11) is a motor protein required for establishing a bipolar spindle in cell division. The role of KIF11 in oral cancer is unclear. Therefore, the present study aimed to assess the role of KIF11 in oral cancer and evaluate its role as a prognostic biomarker and therapeutic target for treating oral cancer. Immunohistochemical analysis demonstrated that KIF11 was expressed in 64 of 99 (64.6%) oral cancer tissues but not in healthy oral epithelia. Strong KIF11 expression was significantly associated with poor prognosis among oral cancer patients (P=0.034), and multivariate analysis confirmed its independent prognostic value. In addition, inhibition of KIF11 expression by transfection of siRNAs into oral cancer cells or treatment of cells with a KIF11 inhibitor significantly suppressed cell proliferation, probably through G2/M arrest and subsequent induction of apoptosis. These results suggest that KIF11 could be a potential prognostic biomarker and therapeutic target for oral cancer. PMID:29115586

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo)

PubMed Central

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-01-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes. PMID:28588392

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells

PubMed Central

The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P.; Cristobal, Alba; Prinsen, Martine B. W.; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J. R.; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

2015-01-01

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/CFZR1 activity as an important determinant in response to CDK4/6-inhibitors. PMID:25562820

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Guo, Wen-jing; Chen, Tong-sheng

2010-02-01

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

Molecular cloning of TA16, a transcriptional repressor that may mediate glucocorticoid-induced growth arrest of leiomyosarcoma cells.

PubMed

Fan, W; Ma, J X; Cheng, L; Norris, J S

1997-08-01

The DDT1 MF2 smooth muscle tumor cell line was derived from an estrogen/androgen-induced leiomyosarcoma that arose in the ductus deferens of a Syrian hamster. The growth of this cell line is arrested at the G0/G1 phase of the cell cycle after treatment with glucocorticoids. To identify the putative gene(s) that are potentially involved in this hormone-induced cell growth arrest, we have used a differential screening technique to clone those genes whose expression is induced or up-regulated by glucocorticoids. A number of glucocorticoid response genes were thereby isolated from the leiomyosarcoma cells. One of these clones, termed TA16, was found to be markedly up-regulated by glucocorticoids in DDT1 MF2 cells, but only marginally changed in GR1 cells, a glucocorticoid-resistant variant that was selected from the wild type DDT1 MF2 cell. Isolation and sequencing of its intact cDNA indicated that the TA16 encodes a protein 485 amino acids long, and its sequence is closely homologous to a novel transcriptional repressor that presumably represses the transcription activity of some zinc finger transcriptional factors through a direct interaction. Transfection assays demonstrated that introduction of an antisense TA16 cDNA expression vector, controlled by an MMTV promoter, into the DDT1 MF2 cell significantly relieved the glucocorticoid-induced cell growth arrest. This finding suggests that TA16 might participate in the mediation of glucocorticoid-induced cell cycle arrest in leiomyosarcoma cells.

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

Essential Roles for Caenorhabditis elegans Lamin Gene in Nuclear Organization, Cell Cycle Progression, and Spatial Organization of Nuclear Pore Complexes

PubMed Central

Liu, Jun; Ben-Shahar, Tom Rolef; Riemer, Dieter; Treinin, Millet; Spann, Perah; Weber, Klaus; Fire, Andrew; Gruenbaum, Yosef

2000-01-01

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs. PMID:11071918

Auxin-Dependent Cell Division and Cell Elongation. 1-Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid Activate Different Pathways1

PubMed Central

Campanoni, Prisca; Nick, Peter

2005-01-01

During exponential phase, the tobacco (Nicotiana tabacum) cell line cv Virginia Bright Italia-0 divides axially to produce linear cell files of distinct polarity. This axial division is controlled by exogenous auxin. We used exponential tobacco cv Virginia Bright Italia-0 cells to dissect early auxin signaling, with cell division and cell elongation as physiological markers. Experiments with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated that these 2 auxin species affect cell division and cell elongation differentially; NAA stimulates cell elongation at concentrations that are much lower than those required to stimulate cell division. In contrast, 2,4-D promotes cell division but not cell elongation. Pertussis toxin, a blocker of heterotrimeric G-proteins, inhibits the stimulation of cell division by 2,4-D but does not affect cell elongation. Aluminum tetrafluoride, an activator of the G-proteins, can induce cell division at NAA concentrations that are not permissive for division and even in the absence of any exogenous auxin. The data are discussed in a model where the two different auxins activate two different pathways for the control of cell division and cell elongation. PMID:15734918

The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

PubMed

Berrak, Özge; Akkoç, Yunus; Arısan, Elif Damla; Çoker-Gürkan, Ajda; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

2016-02-01

Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

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

Suzuki, Kanayo; Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp; Tanaka, Satoshi

2014-01-03

Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDKmore » inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.« less

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer.

PubMed

La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Verrico, Annalisa; Miele, Andrea; Monti, Ludovica; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Ricci, Biancamaria; Soriani, Alessandra; Santoni, Angela; Caraglia, Michele; Porto, Stefania; Da Pozzo, Eleonora; Martini, Claudia; Brancale, Andrea; Marinelli, Luciana; Novellino, Ettore; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Bigogno, Chiara; Dondio, Giulio; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

2015-08-13

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer

PubMed Central

La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Verrico, Annalisa; Miele, Andrea; Monti, Ludovica; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Ricci, Biancamaria; Soriani, Alessandra; Santoni, Angela; Caraglia, Michele; Porto, Stefania; Pozzo, Eleonora Da; Martini, Claudia; Brancale, Andrea; Marinelli, Luciana; Novellino, Ettore; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Bigogno, Chiara; Dondio, Giulio; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

2015-01-01

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethox-yphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4–7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20–50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer. PMID:26132075

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

PubMed

Siede, W; Friedberg, A S; Friedberg, E C

1993-09-01

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

The Cell Cycle Inhibitor p27KIP1: A Key Mediator of G1 Arrest by Androgen Ablation an dby Vitamin D3 Analog

DTIC Science & Technology

2000-02-01

al., 1996; Tyers, 1996). gland . Higher doses of androgen cause growth arrest by p27 increases during differentiation in many cell inducing...innocuous hormones in human prostate cancer patients. These vitamin D3 analogs can inhibit prostate cancer growth and yet they do not cause the negative side...Vitamin D3 and a physiologic does of DHT could cause a synergistic growth arrest in prostate cancer cells (22). The vitamin D3 analogue EB 1089 has the

Oriented cell division: new roles in guiding skin wound repair and regeneration

PubMed Central

Yang, Shaowei; Ma, Kui; Geng, Zhijun; Sun, Xiaoyan; Fu, Xiaobing

2015-01-01

Tissue morphogenesis depends on precise regulation and timely co-ordination of cell division and also on the control of the direction of cell division. Establishment of polarity division axis, correct alignment of the mitotic spindle, segregation of fate determinants equally or unequally between daughter cells, are essential for the realization of oriented cell division. Furthermore, oriented cell division is regulated by intrinsic cues, extrinsic cues and other cues, such as cell geometry and polarity. However, dysregulation of cell division orientation could lead to abnormal tissue development and function. In the present study, we review recent studies on the molecular mechanism of cell division orientation and explain their new roles in skin repair and regeneration. PMID:26582817

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

PubMed

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest

PubMed Central

Gascoyne, Duncan M.; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E.; Croucher, Peter I.; Banham, Alison H.

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology. PMID:26034982

Effect of berberine on cell cycle arrest and cell survival during cerebral ischemia and reperfusion and correlations with p53/cyclin D1 and PI3K/Akt.

PubMed

Chai, Yu-Shuang; Hu, Jun; Lei, Fan; Wang, Yu-Gang; Yuan, Zhi-Yi; Lu, Xi; Wang, Xin-Pei; Du, Feng; Zhang, Dong; Xing, Dong-Ming; Du, Li-Jun

2013-05-15

Berberine acted as a natural medicine with multiple pharmacological activities. In the present study, we examined the effect of berberine against cerebral ischemia damage from cell cycle arrest and cell survival. Oxygen-glucose deprivation of PC12 cells and primary neurons, and carotid artery ligation in mice were used as in vitro and in vivo cerebral ischemia models. We found that the effect of berberine on cell cycle arrest during ischemia was mediated by decreased p53 and cyclin D1, increased phosphorylation of Bad (higher expression of p-Bad and higher ratio of p-Bad to Bad) and decreased cleavage of caspase 3. Meanwhile, berberine activated the PI3K/Akt pathway during the reperfusion, especially the phosphor-activation of Akt, to promote the cell survival. The neural protective effect of berberine was remained in the presence of inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (MEK), but was suppressed by the inhibitors of PI3K and Akt. We demonstrated that berberine induced cell cycle arrest and cell survival to resist cerebral ischemia injury. Copyright © 2013 Elsevier B.V. All rights reserved.

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

PubMed Central

Pervin, Shehla; Singh, Rajan; Chaudhuri, Gautam

2001-01-01

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle. PMID:11248121

Growth arrest and morphological changes triggered by emodin on Trypanosoma cruzi epimastigotes cultivated in axenic medium.

PubMed

De Lima, Ana R; Noris-Suárez, Karem; Bretaña, Antonio; Contreras, Victor T; Navarro, Maria C; Pérez-Ybarra, Luis; Bubis, José

2017-11-01

Emodin is an anthraquinone obtained from Rheum palmatum rootstocks. Here we tested the cytotoxic effects of emodin on Trypanosoma cruzi epimastigotes, as well as the morphological changes that were induced by this compound in the parasite. Emodin was permeable and blocked in vitro cell division of T. cruzi epimastigotes in axenic medium, causing growth arrest in a dose-dependent but reversible manner. Emodin-exposed epimastigotes underwent duplication of organelles, such as the nucleus, kinetoplast and flagellum, but were incapable of completing cytokinesis. Neither elongation of the parasite body nor appearance of the regular longitudinal cleavage furrow was displayed, suggesting that emodin is most likely affecting components of the parasite cytoskeleton. Moreover, drug-treated parasites acquired alterations such as protuberances, folds and indentations on their membrane surface. Since emodin has been shown to be a potent protein kinase CK2 inhibitor, and we have previously described an association between tubulin and CK2 in T. cruzi epimastigotes (De Lima et al. Parasitology132, 511-523, 2006), we also measured the indirect effect of the drug on tubulin. Incubation of epimastigotes with axenic medium containing emodin hindered the endogenous phosphorylation of tubulin in whole-cell parasite extracts. All our results suggested that the parasite CK2 may be important for the maintenance of the morphology and for the regulation of mitosis-cytokinesis transition in T. cruzi epimastigotes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Haplo-Insufficiency of MPK3 in MPK6 Mutant Background Uncovers a Novel Function of These Two MAPKs in Arabidopsis Ovule Development[W

PubMed Central

Wang, Huachun; Liu, Yidong; Bruffett, Kristin; Lee, Justin; Hause, Gerd; Walker, John C.; Zhang, Shuqun

2008-01-01

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage–dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3−/− mpk6+/− plants are fertile. Genetic analysis revealed that the female sterility of mpk3+/− mpk6−/− plants is a sporophytic effect. In mpk3+/− mpk6−/− mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3+/− mpk6−/− ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development. PMID:18364464

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

PubMed Central

Hoeferlin, L. Alexis; Oleinik, Natalia V.; Krupenko, Natalia I.

2011-01-01

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21−/− cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21+/+ cells; such an arrest was not seen in p21-deficient (HCT116 p21−/−) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death. PMID:22593801

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

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.

Modeling Cancer Cell Growth Dynamics In vitro in Response to Antimitotic Drug Treatment

PubMed Central

Lorz, Alexander; Botesteanu, Dana-Adriana; Levy, Doron

2017-01-01

Investigating the role of intrinsic cell heterogeneity emerging from variations in cell-cycle parameters and apoptosis is a crucial step toward better informing drug administration. Antimitotic agents, widely used in chemotherapy, target exclusively proliferative cells and commonly induce a prolonged mitotic arrest followed by cell death via apoptosis. In this paper, we developed a physiologically motivated mathematical framework for describing cancer cell growth dynamics that incorporates the intrinsic heterogeneity in the time individual cells spend in the cell-cycle and apoptosis process. More precisely, our model comprises two age-structured partial differential equations for the proliferative and apoptotic cell compartments and one ordinary differential equation for the quiescent compartment. To reflect the intrinsic cell heterogeneity that governs the growth dynamics, proliferative and apoptotic cells are structured in “age,” i.e., the amount of time remaining to be spent in each respective compartment. In our model, we considered an antimitotic drug whose effect on the cellular dynamics is to induce mitotic arrest, extending the average cell-cycle length. The prolonged mitotic arrest induced by the drug can trigger apoptosis if the time a cell will spend in the cell cycle is greater than the mitotic arrest threshold. We studied the drug’s effect on the long-term cancer cell growth dynamics using different durations of prolonged mitotic arrest induced by the drug. Our numerical simulations suggest that at confluence and in the absence of the drug, quiescence is the long-term asymptotic behavior emerging from the cancer cell growth dynamics. This pattern is maintained in the presence of small increases in the average cell-cycle length. However, intermediate increases in cell-cycle length markedly decrease the total number of cells and can drive the cancer population to extinction. Intriguingly, a large “switch-on/switch-off” increase in the average cell-cycle length maintains an active cell population in the long term, with oscillating numbers of proliferative cells and a relatively constant quiescent cell number. PMID:28913178

Maple polyphenols, ginnalins A-C, induce S- and G2/M-cell cycle arrest in colon and breast cancer cells mediated by decreasing cyclins A and D1 levels.

PubMed

González-Sarrías, Antonio; Ma, Hang; Edmonds, Maxwell E; Seeram, Navindra P

2013-01-15

Polyphenols are bioactive compounds found in plant foods. Ginnalins A-C are polyphenols present in the sap and other parts of the sugar and red maple species which are used to produce maple syrup. Here we evaluated the antiproliferative effects of ginnalins A-C on colon (HCT-116) and breast (MCF-7) tumourigenic and non-tumourigenic colon (CCD-18Co) cells and investigated whether these effects were mediated through cell cycle arrest and/or apoptosis. Ginnalins A-C were twofold more effective against the tumourigenic than non-tumourigenic cells. Among the polyphenols, ginnalin A (84%, HCT-116; 49%, MCF-7) was more effective than ginnalins B and C (50%, HCT-116; 30%, MCF-7) at 50 μM concentrations. Ginnalin A did not induce apoptosis of the cancer cells but arrested cell cycle (in the S- and G(2)/M-phases) and decreased cyclins A and D1 protein levels. These results suggest that maple polyphenols may have potential cancer chemopreventive effects mediated through cell cycle arrest. Copyright © 2012 Elsevier Ltd. All rights reserved.

Phytocalpain controls the proliferation and differentiation fates of cells in plant organ development.

PubMed

Ahn, Joon-Woo; Kim, Moonil; Lim, Jeong Hwa; Kim, Gyung-Tae; Pai, Hyun-Sook

2004-06-01

Calpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During flower development, a single dome-like structure was grown from the flower meristem to generate a large cylinder-shaped flower lacking any floral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.

Discovery and characterization of novel imidazopyridine derivative CHEQ-2 as a potent CDC25 inhibitor and promising anticancer drug candidate.

PubMed

Song, Yu'ning; Lin, Xiaoqian; Kang, Dongwei; Li, Xiao; Zhan, Peng; Liu, Xinyong; Zhang, Qingzhu

2014-07-23

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation via the interactions with CDK/Cyclin complexes. Overexpression of CDC25 proteins is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, inhibiting CDC25 activity in cancer treatment appears a good therapeutic strategy. In this article, refinement of the initial hit XDW-1 by synthesis and screening of a focused compound library led to the identification of a novel set of imidazopyridine derivatives as potent CDC25 inhibitors. Among them, the most potent molecule was CHEQ-2, which could efficiently inhibit the activities of CDC25A/B enzymes as well as the proliferation of various different types of cancer cell lines in vitro assay. Moreover, CHEQ-2 triggered S-phase cell cycle arrest in MCF-7, HepG2 and HT-29 cell lines, accompanied by generation of ROS, mitochondrial dysfunction and apoptosis. Besides, oral administration of CHEQ-2 (10 mg/kg) significantly inhibited xenografted human liver tumor growth in nude mice, while demonstrated extremely low toxicity (LD50 > 2000 mg/kg). These findings make CHEQ-2 a good starting point for further investigation and structure modification. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Embryology of Cardiopteris (Cardiopteridaceae, Aquifoliales), with emphasis on unusual ovule and seed development.

PubMed

Tobe, Hiroshi

2016-09-01

Cardiopteris (Cardiopteridaceae), a twining herb of two or three species distributed from Southeast Asia to Northern Australia, requires an embryological study for better understanding of its reproductive features. The present study of C. quinqueloba showed that the ovule and seed development involves a number of unusual structures, most of which are unknown elsewhere in angiosperms. The ovule pendant from the apical placenta is straight (not orthotropous), ategmic, and tenuinucellate, developing a monosporic seven-celled/eight-nucleate female gametophyte with an egg apparatus on the funicular side. Fertilization occurs by a pollen tube entering from the funicular side, resulting in a zygote on the funicular side. The endosperm is formed by the cell on the funicular side in the two endosperm cell stage. While retaining a (pro)embryo/endosperm as it is, the raphe (differentiating late in pre-fertilization stages) elongates toward the antiraphal side during post-fertilization stages, resulting in an anatropous seed. The two-cell-layered nucellar epidermis (belatedly forming by periclinal divisions), along with the raphe, envelops the embryo/endosperm entirely as the seed coat. The possibility was discussed that the arrested integument development triggers a series of the subsequent unusual structures of ovule and seed development. The fertilization mode in Cardiopteris underpins the hypothesis that the Polygonum‒type female gametophyte comprises two four-celled archegonia.

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

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

PubMed

Park, Heon Joo; Lee, Sang Hwa; Chung, HyunSook; Rhee, Yun Hee; Lim, Byung Uk; Ha, Sung Whan; Griffin, Robert J; Lee, Hyung Sik; Song, Chang Won; Choi, Eun Kyung

2003-01-01

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Characterization of mitochondrial injury after cardiac arrest (COMICA).

PubMed

Donnino, Michael W; Liu, Xiaowen; Andersen, Lars W; Rittenberger, Jon C; Abella, Benjamin S; Gaieski, David F; Ornato, Joseph P; Gazmuri, Raúl J; Grossestreuer, Anne V; Cocchi, Michael N; Abbate, Antonio; Uber, Amy; Clore, John; Peberdy, Mary Anne; Callaway, Clifton W

2017-04-01

Mitochondrial injury post-cardiac arrest has been described in pre-clinical settings but the extent to which this injury occurs in humans remains largely unknown. We hypothesized that increased levels of mitochondrial biomarkers would be associated with mortality and neurological morbidity in post-cardiac arrest subjects. We performed a prospective multicenter study of post-cardiac arrest subjects. Inclusion criteria were comatose adults who suffered an out-of-hospital cardiac arrest. Mitochondrial biomarkers were measured at 0, 12, 24, 36 and 48h after return of spontaneous circulation as well as in healthy controls. Out of 111 subjects enrolled, 102 had evaluable samples at 0h. Cardiac arrest subjects had higher baseline cytochrome c levels compared to controls (2.18ng/mL [0.74, 7.74] vs. 0.16ng/mL [0.03, 0.91], p<0.001), and subjects who died had higher 0h cytochrome c levels compared to survivors (3.66ng/mL [1.40, 14.9] vs. 1.27ng/mL [0.16, 2.37], p<0.001). There were significantly higher Ribonuclease P (RNaseP) (3.3 [1.2, 5.7] vs. 1.2 [0.8, 1.2], p<0.001) and Beta-2microglobulin (B2M) (12.0 [1.0, 22.9], vs. 0.6 [0.6, 1.3], p<0.001) levels in cardiac arrest subjects at baseline compared to the control subjects. There were no differences between survivors and non-survivors for mitochondrial DNA, nuclear DNA, or cell free DNA. Cytochrome c was increased in post- cardiac arrest subjects compared to controls, and in post-cardiac arrest non-survivors compared to survivors. Nuclear DNA and cell free DNA was increased in plasma of post-cardiac arrest subjects. There were no differences in mitochondrial DNA, nuclear DNA, or cell free DNA between survivors and non-survivors. Mitochondrial injury markers showed mixed results in the post-cardiac arrest period. Future research needs to investigate these differences. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Mitochondrial Injury after Cardiac Arrest (COMICA)

PubMed Central

Donnino, Michael W.; Liu, Xiaowen; Andersen, Lars W.; Rittenberger, Jon C.; Abella, Benjamin S.; Gaieski, David F.; Ornato, Joseph P.; Gazmuri, Raúl J.; Grossestreur, Anne V.; Cocchi, Michaen N.; Abbate, Antonio; Uber, Amy; Clore, John; Peberdy, Mary Anne; Callaway, Clifton

2017-01-01

Introduction Mitochondrial injury post-cardiac arrest has been described in pre-clinical settings but the extent to which this injury occurs in humans remains largely unknown. We hypothesized that increased levels of mitochondrial biomarkers would be associated with mortality and neurological morbidity in post-cardiac arrest subjects. Methods We performed a prospective multicenter study of post-cardiac arrest subjects. Inclusion criteria were comatose adults who suffered an out-of-hospital cardiac arrest. Mitochondrial biomarkers were measured at 0, 12, 24, 36 and 48 hours after return of spontaneous circulation as well as in healthy controls. Results Out of 111 subjects enrolled, 102 had evaluable samples at 0 hours. Cardiac arrest subjects had higher baseline cytochrome c levels compared to controls (2.18 ng/mL [0.74, 7.74] vs. 0.16 ng/mL [0.03, 0.91], p<0.001), and subjects who died had higher 0 hours cytochrome c levels compared to survivors (3.66 ng/mL [1.40, 14.9] vs. 1.27 ng/mL [0.16, 2.37], p<0.001). There were significantly higher RNAase P (3.3 [1.2, 5.7] vs. 1.2 [0.8, 1.2], p<0.001) and B2M (12.0 [1.0, 22.9], vs. 0.6 [0.6, 1.3], p<0.001) levels in cardiac arrest subjects at baseline compared to the control subjects. There were no differences between survivors and non-survivors for mitochondrial DNA, nuclear DNA, or cell free DNA. Conclusions Cytochrome C was increased in post-cardiac arrest subjects compared to controls, and in post-cardiac arrest non-survivors compared to survivors. Nuclear DNA and cell free DNA was increased in plasma of post-cardiac arrest subjects. There were no differences in mitochondrial DNA, nuclear DNA, or cell free DNA between survivors and non-survivors. Mitochondrial injury markers showed mixed results in post-arrest period. Future research needs to investigate these differences. PMID:28126408

Alternative role of HuD splicing variants in neuronal differentiation.

PubMed

Hayashi, Satoru; Yano, Masato; Igarashi, Mana; Okano, Hirotaka James; Okano, Hideyuki

2015-03-01

HuD is a neuronal RNA-binding protein that plays an important role in neuronal differentiation of the nervous system. HuD has been reported to have three RNA recognition motifs (RRMs) and three splice variants (SVs) that differ in their amino acid sequences between RRM2 and RRM3. This study investigates whether these SVs have specific roles in neuronal differentiation. In primary neural epithelial cells under differentiating conditions, HuD splice variant 1 (HuD-sv1), which is a general form, and HuD-sv2 were expressed at all tested times, whereas HuD-sv4 was transiently expressed at the beginning of differentiation, indicating that HuD-sv4 might play a role compared different from that of HuD-sv1. Indeed, HuD-sv4 did not promote neuronal differentiation in epithelial cells, whereas HuD-sv1 did promote neuronal differentiation. HuD-sv4 overexpression showed less neurite-inducing activity than HuD-sv1 in mouse neuroblastoma N1E-115 cells; however, HuD-sv4 showed stronger growth-arresting activity. HuD-sv1 was localized only in the cytoplasm, whereas HuD-sv4 was localized in both the cytoplasm and the nuclei. The Hu protein has been reported to be involved in translation and alternative splicing in the cytoplasm and nuclei, respectively. Consistent with this observation, HuD-sv1 showed translational activity on p21, which plays a role in growth arrest and neuronal differentiation, whereas HuD-sv4 did not. By contrast, HuD-sv4 showed stronger pre-mRNA splicing activity than did HuD-sv1 on Clasp2, which participates in cell division. Therefore, HuD SVs might play a role in controlling the timing of proliferation/differentiation switching by controlling the translation and alternative splicing of target genes. © 2014 Wiley Periodicals, Inc.

The Caenorhabditis elegans gene ham-1 regulates daughter cell size asymmetry primarily in divisions that produce a small anterior daughter cell

PubMed Central

Kovacevic, Ismar; Bao, Zhirong

2018-01-01

C. elegans cell divisions that produce an apoptotic daughter cell exhibit Daughter Cell Size Asymmetry (DCSA), producing a larger surviving daughter cell and a smaller daughter cell fated to die. Genetic screens for mutants with defects in apoptosis identified several genes that are also required for the ability of these divisions to produce daughter cells that differ in size. One of these genes, ham-1, encodes a putative transcription factor that regulates a subset of the asymmetric cell divisions that produce an apoptotic daughter cell. In a survey of C. elegans divisions, we found that ham-1 mutations affect primarily anterior/posterior divisions that produce a small anterior daughter cell. The affected divisions include those that generate an apoptotic cell as well as those that generate two surviving cells. Our findings suggest that HAM-1 primarily promotes DCSA in a certain class of asymmetric divisions. PMID:29668718

Stationary Size Distributions of Growing Cells with Binary and Multiple Cell Division

NASA Astrophysics Data System (ADS)

Rading, M. M.; Engel, T. A.; Lipowsky, R.; Valleriani, A.

2011-10-01

Populations of unicellular organisms that grow under constant environmental conditions are considered theoretically. The size distribution of these cells is calculated analytically, both for the usual process of binary division, in which one mother cell produces always two daughter cells, and for the more complex process of multiple division, in which one mother cell can produce 2 n daughter cells with n=1,2,3,… . The latter mode of division is inspired by the unicellular algae Chlamydomonas reinhardtii. The uniform response of the whole population to different environmental conditions is encoded in the individual rates of growth and division of the cells. The analytical treatment of the problem is based on size-dependent rules for cell growth and stochastic transition processes for cell division. The comparison between binary and multiple division shows that these different division processes lead to qualitatively different results for the size distribution and the population growth rates.

Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

PubMed

Shen, Ziying; Ma, Yunqing; Ji, Zhonghao; Hao, Yang; Yan, Xuan; Zhong, Yuan; Tang, Xiaochun; Ren, Wenzhi

2018-02-09

Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.

Radiotherapy induces cell cycle arrest and cell apoptosis in nasopharyngeal carcinoma via the ATM and Smad pathways.

PubMed

Li, Ming-Yi; Liu, Jin-Quan; Chen, Dong-Ping; Li, Zhou-Yu; Qi, Bin; He, Lu; Yu, Yi; Yin, Wen-Jin; Wang, Meng-Yao; Lin, Ling

2017-09-02

Nasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck which is harmful to human's health. Radiotherapy is commonly used in the treatment of NPC and it induces immediate cell cycle arrest and cell apoptosis. However, the mechanism remains unknown. Evidences suggested the activation of Ataxia telangiectasia mutated (ATM) pathway and Smad pathway are 2 of the important crucial mediators in the function of radiotherapy. In this study, we performed in vitro assays with human nasopharyngeal carcinoma CNE-2 cells and in vivo assays with nude mice to investigate the role of the ATM and Smad pathways in the treatment of nasopharyngeal carcinoma with radiotherapy. The results suggested that radiation induced activation of ATM pathway by inducing expression of p-ATM, p-CHK1, p-CHK2, p15 and inhibiting expression of p-Smad3. In addition, Caspase3 expression was increased while CDC25A was decreased, leading to cell cycle arrest and cell apoptosis. On the other hand, activation of Smad3 can inhibited the ATM pathway and attenuated the efficacy of radiation. In summary, we suggest that both ATM and Smad pathways contribute to the cell cycle arrest and cell apoptosis during nasopharyngeal carcinoma cells treated with radiation.

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.

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells.

PubMed

Yen, Andrew; Varvayanis, Susi; Smith, James L; Lamkin, Thomas J

2006-02-01

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

A novel anticancer agent, decursin, induces G1 arrest and apoptosis in human prostate carcinoma cells.

PubMed

Yim, Dongsool; Singh, Rana P; Agarwal, Chapla; Lee, Sookyeon; Chi, Hyungjoon; Agarwal, Rajesh

2005-02-01

We isolated a coumarin compound decursin (C(19)H(20)O(5); molecular weight 328) from Korean angelica (Angelica gigas) root and characterized it by spectroscopy. Here, for the first time, we observed that decursin (25-100 micromol/L) treatment for 24 to 96 hours strongly inhibits growth and induces death in human prostate carcinoma DU145, PC-3, and LNCaP cells. Furthermore, we observed that decursinol [where (CH(3))(2)-C=CH-COO- side chain of decursin is substituted with -OH] has much lower effects compared with decursin, suggesting a possible structure-activity relationship. Decursin-induced growth inhibition was associated with a strong G(1) arrest (P < 0.001) in DU145 and LNCaP cells, and G(1), S as well as G(2)-M arrests depending upon doses and treatment times in PC-3 cells. Comparatively, decursin was nontoxic to human prostate epithelial PWR-1E cells and showed only moderate growth inhibition and G(1) arrest. Consistent with G(1) arrest in DU145 cells, decursin strongly increased protein levels of Cip1/p21 but showed a moderate increase in Kip1/p27 with a decrease in cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E kinase activity, and increased binding of CDK inhibitor (CDKI) with CDK. Decursin-caused cell death was associated with an increase in apoptosis (P < 0.05-0.001) and cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase; however, pretreatment with all-caspases inhibitor (z-VAD-fmk) only partially reversed decursin-induced apoptosis, suggesting the involvement of both caspase-dependent and caspase-independent pathways. These findings suggest the novel anticancer efficacy of decursin mediated via induction of cell cycle arrest and apoptosis selectively in human prostate carcinoma cells.

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

PubMed

Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

2014-01-01

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Evodiamine Induces Cell Growth Arrest, Apoptosis and Suppresses Tumorigenesis in Human Urothelial Cell Carcinoma Cells.

PubMed

Shi, Chung-Sheng; Li, Jhy-Ming; Chin, Chih-Chien; Kuo, Yi-Hung; Lee, Ying-Ray; Huang, Yun-Ching

2017-03-01

Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities including vasodilatation, analgesia, anti-cardiovascular disease, anti-Alzheimer's disease, anti-inflammation, and anti-tumor activity. This study analyzes the anti-tumor effects of evodiamine on cellular growth, tumorigenesis, cell cycle and apoptosis induction of human urothelial cell carcinoma (UCC) cells. The present study showed that evodiamine significantly inhibited the proliferation of UCC cells in a dose- and time-dependent manner. Also, evodiamine suppressed the tumorigenesis of UCC cells in vitro. Moreover, evodiamine caused G 2 /M cell-cycle arrest and induced caspase-dependent apoptosis in UCC cells. Finally, we demonstrated that evodiamine exhibits better cytotoxic than 5-fluorouracil, a clinical chemotherapeutic drug, for UCC cells. Evodiamine induces growth inhibition, tumorigenesis suppression, cell-cycle arrest, and apoptosis induction in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC cells and is worthy for further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

Galán-Malo, Patricia; Vela, Laura; Gonzalo, Oscar

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x{sub L} and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x{sub L} gene was silenced in A549 cells, vincristinemore » induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x{sub L} or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x{sub L} switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x{sub L}/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types. Highlights: ► Vincristine induces cell death by apoptosis or mitotic catastrophe. ► Apoptosis-proficient cells die by apoptosis during mitosis upon vincristine treatment. ► p53wt apoptosis-deficient cells undergo apoptosis from a G1-like tetraploid state. ► p53mt apoptosis-deficient cells can survive and divide giving rise to 8N cells.« less

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.

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

PubMed

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

2014-03-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. Copyright © 2014. Published by Elsevier Inc.

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube

PubMed Central

Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Gole, Christophe; Barresi, Michael J.F.

2014-01-01

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226x delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube. PMID:24370453

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

Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases.

PubMed

Abraham, R T; Acquarone, M; Andersen, A; Asensi, A; Bellé, R; Berger, F; Bergounioux, C; Brunn, G; Buquet-Fagot, C; Fagot, D

1995-01-01

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.

The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

NASA Technical Reports Server (NTRS)

Hu, B.; Zhou, X. Y.; Wang, X.; Zeng, Z. C.; Iliakis, G.; Wang, Y.

2001-01-01

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

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

Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp; Yamauchi, Motohiro; Oka, Yasuyoshi

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% andmore » 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.« less

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc- cells

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.

1994-01-01

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the -49 lymphoma variant (cyc-) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1 (dmPGA1) inhibits DNA synthesis and cell growth in cyc- cells. DNA synthesis is inhibited 42% by dmPGA1 (50 microM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the alpha, beta unsaturated ketone ring. Dimethyl PGA1 is most effective in inhibiting DNA synthesis in cyc- cells, with prostaglandins PGE1 and PGB1 being less potent inhibitors of DNA synthesis. DmPGE2 caused a significant stimulation of DNA synthesis. S-49 cyc- variant cells exposed to (30-50 microns) dmPGA1, arrested in the G1 phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc- cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G1 cell cycle block.(ABSTRACT TRUNCATED AT 250 WORDS).

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

Antagonism between curcumin and the topoisomerase II inhibitor etoposide

PubMed Central

Saleh, Ekram M.; El-awady, Raafat A; Eissa, Nadia A.; Abdel-Rahman, Wael M.

2012-01-01

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program. PMID:22895066

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

PubMed

Orsi, Guillermo A; Joyce, Eric F; Couble, Pierre; McKim, Kim S; Loppin, Benjamin

2010-10-15

The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

Effector-Triggered Self-Replication in Coupled Subsystems.

PubMed

Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

2017-11-13

In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ectodermal fragments from normal frog gastrulae condition substrata to support normal and hybrid mesodermal cell migration in vitro.

PubMed

Nakatsuji, N; Johnson, K E

1984-06-01

Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers during gastrulation. They can also be deposited in vitro where they condition substrata in such a way that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos can condition culture substrata so as to promote moderate cell attachment and, for one particular gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested hybrid embryos. These results provide new evidence to support the hypothesis that extracellular fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.

Mutation of the Membrane-Associated M1 Protease APM1 Results in Distinct Embryonic and Seedling Developmental Defects in Arabidopsis[C][W

PubMed Central

Peer, Wendy Ann; Hosein, Fazeeda N.; Bandyopadhyay, Anindita; Makam, Srinivas N.; Otegui, Marisa S.; Lee, Gil-Je; Blakeslee, Joshua J.; Cheng, Yan; Titapiwatanakun, Boosaree; Yakubov, Bahktiyor; Bangari, Bharat; Murphy, Angus S.

2009-01-01

Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A–sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA. PMID:19531600

Testicular feminization in the Finnish racoon dog (Nyctereutes procyonoides).

PubMed

Smith, A; Nes, N; Berg, K A; Valtonen, M; Mäkinen, A; Lukola, A

1983-12-01

The clinical features of testicular feminization in the racoon dog (Nyctereutes procyonoides) are reported. The condition is characterized by a normal male karyotype, but a mixed phenotype consisting of vulva, enlarged clitoris and scrotal testes. Partial spermatogenesis with a relative arrest at the first meiotic division was observed. The likely underlying genetic defect and mode of inheritance are discussed, together with implications for breeding programmes.

RhoA-stimulated intra-capillary morphology switch facilitates the arrest of individual circulating tumor cells.

PubMed

Huang, Xi; Yang, Yu; Zhao, Yuwei; Cao, Dan; Ai, Xiaolin; Zeng, Anqi; Gou, Maling; Cai, Lulu; Yang, Hanshuo; Zhao, Chengjian

2018-05-15

Metastasis is the primary cause of death for most cancer patients. Hematogenous arrest of circulating tumor cells (CTCs) is an essential prerequisite for metastases formation. Using transparent transgenic zebrafish (kdrl:eGFP; Casper), together with resonant laser scanning confocal microscopy, we tracked the fate of CTCs in vivo in the blood circulation for days. We found the intra-capillary morphology-switch (ICMS) of individual CTCs from strip to sphere was necessary for their intravascular arrests. Further genetic and pharmacological inhibition experiments indicated that the RhoA signaling was necessary for ICMS and the arrest of CTCs. At last, we demonstrated that early treatment by a clinically approved RhoA/ROCK inhibitor, Fasudil, could efficiently inhibit the initial arrest of individual CTCs and reduce the incidence of tumor metastasis in both zebrafish and mouse models. These results together indicate that RhoA-stimulated ICMS represents a mechanism for the arrest of individual CTCs, providing a potential target for future treatments of hematogenous metastatic disease. © 2017 UICC.

Distinct prophase arrest mechanisms in human male meiosis.

PubMed

Jan, Sabrina Z; Jongejan, Aldo; Korver, Cindy M; van Daalen, Saskia K M; van Pelt, Ans M M; Repping, Sjoerd; Hamer, Geert

2018-04-16

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development. © 2018. Published by The Company of Biologists Ltd.

Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells

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

Su, Miaoxian; Chung, Hau Yin, E-mail: anthonychung@cuhk.edu.hk; Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR

2011-07-29

Highlights: {yields} Deoxyelephantopin (ESD) inhibited cell proliferation in the human nasopharyngeal cancer CNE cells. {yields} ESD induced cell cycle arrest in S and G2/M phases via modulation of cell cycle regulatory proteins. {yields} ESD triggered apoptosis by dysfunction of mitochondria and induction of both intrinsic and extrinsic apoptotic signaling pathways. {yields} ESD also triggered Akt, ERK, and JNK signaling pathways. -- Abstract: Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cellsmore » are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential ({Delta}{Psi}m), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).« less

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

Renal denervation prevents long-term sequelae of ischemic renal injury

PubMed Central

Kim, Jinu; Padanilam, Babu J.

2014-01-01

Signals that drive interstitial fibrogenesis after renal ischemia reperfusion injury remain undefined. Sympathetic activation is manifest even in the early clinical stages of chronic kidney disease and is directly related to disease severity. A role for renal nerves in renal interstitial fibrogenesis in the setting of ischemia reperfusion injury has not been studied. In male 129S1/SvImJ mice, ischemia reperfusion injury induced tubulointerstitial fibrosis as indicated by collagen deposition and profibrotic protein expression 4 to 16 days after the injury.. Leukocyte influx, proinflammatory protein expression, oxidative stress, apoptosis, and cell cycle arrest at G2/M phase were enhanced after ischemia reperfusion injury. Renal denervation at the time of injury or up to 1 day post-injury improved histology, decreased proinflammatory/profibrotic responses and apoptosis, and prevented G2/M cell cycle arrest in the kidney. Treatment with afferent nerve-derived calcitonin gene-related peptide (CGRP) or efferent nerve-derived norepinephrine in denervated and ischemia reperfusion injury-induced kidneys mimicked innervation, restored inflammation and fibrosis, induced G2/M arrest, and enhanced TGF-β1 activation. Blocking norepinephrine or CGRP function using respective receptor blockers prevented these effects. Consistent with the in vivo study, treatment with either norepinephrine or CGRP induced G2/M cell cycle arrest in HK-2 proximal tubule cells, whereas antagonists against their respective receptors prevented G2/M arrest. Thus, renal nerve stimulation is a primary mechanism and renal nerve-derived factors drive epithelial cell cycle arrest and the inflammatory cascade causing interstitial fibrogenesis after ischemia reperfusion injury. PMID:25207878

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

PubMed Central

Balbach, Sebastian Thomas; Esteves, Telma Cristina; Houghton, Franchesca Dawn; Siatkowski, Marcin; Pfeiffer, Martin Johannes; Tsurumi, Chizuko; Kanzler, Benoit; Fuellen, Georg; Boiani, Michele

2012-01-01

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development. PMID:22530006

[The role of cellular senescence in carcinogenesis and antitumor therapy].

PubMed

Mosieniak, Grazyna; Strzeszewska, Anna

2014-01-01

Cellular senescence is the process that lead to terminal growth arrest induced by unrepairable double strand DNA damage (DSB). Moreover, activation of the oncogenes as well as inhibition of the tumor suppressor genes were shown to contribute to senescence induction and the senescent cells were identified in the premalignant lesions. Thus senescence is considered as an natural antitumor barrier that act at the early stages of cancerogenesis to stop the proliferation of transformed cells. Interestingly, the premalignant cells that escaped senescence and progress into full blown tumor cells still remain sensitive to induction of senescence, for example during chemio- or radiotherapy. Thus, induction of cancer cell senescence, similarly to apoptosis, is considered to restrain tumor growth and thus contribute to effectiveness of anticancer therapy. The senescent cells, although do not proliferate, remain viable and metabolically active. They secret a lot of cytokines, mitogens as well as enzymes degrading extracellular matrix. These factors can have opposing effect on neighboring cells, leading to senescence induction or stimulation of proliferation. Thus, senescence can act as an double edge sword that inhibit the propagation of potentially dangerous, transformed cells on one hand or induce cell division of the same cell on the other. Presently a lot of work is focused on finding new therapeutic strategies that would involve the tumor targeted senescence induction in both early late stages of cancer development. Nevertheless, the unwanted influence of the senescent cells on the microenvironment, requires careful monitoring the effects of pro-senescent therapies in each case.

Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

PubMed

Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

2017-03-01

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

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

Magalhães, Hemerson I.F.; Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Paraíba; Wilke, Diego V.

2013-10-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation,more » loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.« less

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine Dependent Arrest in the T Cell Signaling Cascade

PubMed Central

Kelleher, Raymond J.; Balu-Iyer, Sathy; Loyall, Jenni; Sacca, Anthony J.; Shenoy, Gautam N.; Peng, Peng; Iyer, Vandana; Fathallah, Anas M.; Berenson, Charles S.; Wallace, Paul K.; Tario, Joseph; Odunsi, Kunle; Bankert, Richard B.

2015-01-01

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients’ anti-tumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80nm. The T cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immune suppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T cell function represent a potential therapeutic target for patients with ovarian cancer. PMID:26112921

Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.

PubMed

Jaiswal, Aruna S; Marlow, Benjamin P; Gupta, Nirupama; Narayan, Satya

2002-12-05

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

Automated cell tracking identifies mechanically oriented cell divisions during Drosophila axis elongation.

PubMed

Wang, Michael F Z; Hunter, Miranda V; Wang, Gang; McFaul, Christopher; Yip, Christopher M; Fernandez-Gonzalez, Rodrigo

2017-04-01

Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo. © 2017. Published by The Company of Biologists Ltd.

Concerted control of Escherichia coli cell division

PubMed Central

Osella, Matteo; Nugent, Eileen; Cosentino Lagomarsino, Marco

2014-01-01

The coordination of cell growth and division is a long-standing problem in biology. Focusing on Escherichia coli in steady growth, we quantify cell division control using a stochastic model, by inferring the division rate as a function of the observable parameters from large empirical datasets of dividing cells. We find that (i) cells have mechanisms to control their size, (ii) size control is effected by changes in the doubling time, rather than in the single-cell elongation rate, (iii) the division rate increases steeply with cell size for small cells, and saturates for larger cells. Importantly, (iv) the current size is not the only variable controlling cell division, but the time spent in the cell cycle appears to play a role, and (v) common tests of cell size control may fail when such concerted control is in place. Our analysis illustrates the mechanisms of cell division control in E. coli. The phenomenological framework presented is sufficiently general to be widely applicable and opens the way for rigorous tests of molecular cell-cycle models. PMID:24550446

Ribonuclease E modulation of the bacterial SOS response.

PubMed

Manasherob, Robert; Miller, Christine; Kim, Kwang-sun; Cohen, Stanley N

2012-01-01

Plants, animals, bacteria, and Archaea all have evolved mechanisms to cope with environmental or cellular stress. Bacterial cells respond to the stress of DNA damage by activation of the SOS response, the canonical RecA/LexA-dependent signal transduction pathway that transcriptionally derepresses a multiplicity of genes-leading to transient arrest of cell division and initiation of DNA repair. Here we report the previously unsuspected role of E. coli endoribonuclease RNase E in regulation of the SOS response. We show that RNase E deletion or inactivation of temperature-sensitive RNase E protein precludes normal initiation of SOS. The ability of RNase E to regulate SOS is dynamic, as down regulation of RNase E following DNA damage by mitomycin C resulted in SOS termination and restoration of RNase E function leads to resumption of a previously aborted response. Overexpression of the RraA protein, which binds to the C-terminal region of RNase E and modulates the actions of degradosomes, recapitulated the effects of RNase E deficiency. Possible mechanisms for RNase E effects on SOS are discussed.

Ribonuclease E Modulation of the Bacterial SOS Response

PubMed Central

Manasherob, Robert; Miller, Christine; Kim, Kwang-sun; Cohen, Stanley N.

2012-01-01

Plants, animals, bacteria, and Archaea all have evolved mechanisms to cope with environmental or cellular stress. Bacterial cells respond to the stress of DNA damage by activation of the SOS response, the canonical RecA/LexA-dependent signal transduction pathway that transcriptionally derepresses a multiplicity of genes–leading to transient arrest of cell division and initiation of DNA repair. Here we report the previously unsuspected role of E. coli endoribonuclease RNase E in regulation of the SOS response. We show that RNase E deletion or inactivation of temperature-sensitive RNase E protein precludes normal initiation of SOS. The ability of RNase E to regulate SOS is dynamic, as down regulation of RNase E following DNA damage by mitomycin C resulted in SOS termination and restoration of RNase E function leads to resumption of a previously aborted response. Overexpression of the RraA protein, which binds to the C-terminal region of RNase E and modulates the actions of degradosomes, recapitulated the effects of RNase E deficiency. Possible mechanisms for RNase E effects on SOS are discussed. PMID:22719885

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation

PubMed Central

Lunt, Sophia Y.; Muralidhar, Vinayak; Hosios, Aaron M.; Israelsen, William J.; Gui, Dan Y.; Newhouse, Lauren; Ogrodzinski, Martin; Hecht, Vivian; Xu, Kali; Acevedo, Paula N. Marín; Hollern, Daniel P.; Bellinger, Gary; Dayton, Talya L.; Christen, Stefan; Elia, Ilaria; Dinh, Anh T.; Stephanopoulos, Gregory; Manalis, Scott R.; Yaffe, Michael B.; Andrechek, Eran R.; Fendt, Sarah-Maria; Heiden, Matthew G. Vander

2014-01-01

SUMMARY Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2-deletion affects proliferation and metabolism in non-transformed, non-immortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis. PMID:25482511

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

PubMed

Havas, Aaron P; Rodrigues, Kameron B; Bhakta, Anvi; Demirjian, Joseph A; Hahn, Seongmin; Tran, Jack; Scavello, Margarethakay; Tula-Sanchez, Ana A; Zeng, Yi; Schmelz, Monika; Smith, Catharine L

2016-12-01

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of β-tubulin.

PubMed

Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong; Kim, Su-Hyeong; Marcus, Adam I; Kumari, Vandana; Ji, Xinhua; Yang, Zhen; Vowell, Courtney L; Wipf, Peter; Uechi, Guy T; Yates, Nathan A; Romero, Guillermo; Sarkar, Saumendra N; Singh, Shivendra V

2014-01-17

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.

Simulated-microgravity induced G2/M arrest in zebrafish embryonic cell is regulated by dre-miR-22a and its target cep135

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Microgravity has been recognized as a major environmental factor that can induce a number of adverse effects such as bone loss, skeletal muscle atrophy, cardiovascular problems and immune system dysregulation, etc. The underlying mechanisms are not absolutely identified yet. Our previous study demonstrated centrosomal protein of 135 kDa (CEP135) might be a microgravity sensitive molecule. In this study, the expression and regulation of CEP135 and its possible roles in cell cycle regulation under simulated microgravity (SMG) condition were investigated. SMG can induce significant increasing of cep135 in zebrafish embryos, detected by both in situ hybridization and RT-qPCR, while CEP135 protein level was significantly decreased, tested by western blot. The similar results were also obtained in zebrafish embryonic cells (ZF4) exposed to SMG. Accordingly, the expression level of dre-miR-22a, which might be the potential miRNA for targeting cep135, was significantly increased in SMG exposed ZF4 cells. By combining the results obtained from transfection and dual luciferase reporter assay, we firstly confirmed that dre-miR-22a regulated the expression of cep135 in ZF4 cells. Further investigation on cell cycle demonstrated SMG induced a significant arrest in G2/M phase. Transfection of dre-miR-22a also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells is via cep135, while dre-miR-22a plays a key role in modulating this effect. Key Words: Simulated-microgravity; cep135; dre-miR-22a; G2/M arrest; zebrafish embryonic cell

The C. elegans engrailed homolog ceh-16 regulates the self-renewal expansion division of stem cell-like seam cells.

PubMed

Huang, Xinxin; Tian, E; Xu, Yanhua; Zhang, Hong

2009-09-15

Stem cells undergo symmetric and asymmetric division to maintain the dynamic equilibrium of the stem cell pool and also to generate a variety of differentiated cells. The homeostatic mechanism controlling the choice between self-renewal and differentiation of stem cells is poorly understood. We show here that ceh-16, encoding the C. elegans ortholog of the transcription factor Engrailed, controls symmetric and asymmetric division of stem cell-like seam cells. Loss of function of ceh-16 causes certain seam cells, which normally undergo symmetric self-renewal expansion division with both daughters adopting the seam cell fate, to divide asymmetrically with only one daughter retaining the seam cell fate. The human engrailed homolog En2 functionally substitutes the role of ceh-16 in promoting self-renewal expansion division of seam cells. Loss of function of apr-1, encoding the C. elegans homolog of the Wnt signaling component APC, results in transformation of self-renewal maintenance seam cell division to self-renewal expansion division, leading to seam cell hyperplasia. The apr-1 mutation suppresses the seam cell division defect in ceh-16 mutants. Our study reveals that ceh-16 interacts with the Wnt signaling pathway to control the choice between self-renewal expansion and maintenance division and also demonstrates an evolutionarily conserved function of engrailed in promoting cell proliferation.

Elongator complex is critical for cell cycle progression and leaf patterning in Arabidopsis.

PubMed

Xu, Deyang; Huang, Weihua; Li, Yang; Wang, Hua; Huang, Hai; Cui, Xiaofeng

2012-03-01

The mitotic cell cycle in higher eukaryotes is of pivotal importance for organ growth and development. Here, we report that Elongator, an evolutionarily conserved histone acetyltransferase complex, acts as an important regulator of mitotic cell cycle to promote leaf patterning in Arabidopsis. Mutations in genes encoding Elongator subunits resulted in aberrant cell cycle progression, and the altered cell division affects leaf polarity formation. The defective cell cycle progression is caused by aberrant DNA replication and increased DNA damage, which activate the DNA replication checkpoint to arrest the cell cycle. Elongator interacts with proliferating cell nuclear antigen (PCNA) and is required for efficient histone 3 (H3) and H4 acetylation coupled with DNA replication. Levels of chromatin-bound H3K56Ac and H4K5Ac known to associate with replicons during DNA replication were reduced in the mutants of both Elongator and chromatin assembly factor 1 (CAF-1), another protein complex that physically interacts with PCNA for DNA replication-coupled chromatin assembly. Disruptions of CAF-1 also led to severe leaf polarity defects, which indicated that Elongator and CAF-1 act, at least partially, in the same pathway to promote cell cycle progression. Collectively, our results demonstrate that Elongator is an important regulator of mitotic cell cycle, and the Elongator pathway plays critical roles in promoting leaf polarity formation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

PubMed Central

Western, Patrick S.; Ralli, Rachael A.; Wakeling, Stephanie I.; Lo, Camden; van den Bergen, Jocelyn A.; Miles, Denise C.; Sinclair, Andrew H.

2011-01-01

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility. PMID:21674058

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

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

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

PubMed

Stępiński, Dariusz

2016-08-01

Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

PubMed

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa E; Cuervo, Patricia; Jesus, José Batista de

2017-10-01

Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

PubMed Central

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa e; Cuervo, Patricia; de Jesus, José Batista

2017-01-01

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis. PMID:28953994

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

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

Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562more » cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.« less

Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae.

PubMed

Kumar, Ravinder; Dhali, Snigdha; Srikanth, Rapole; Ghosh, Santanu Kumar; Srivastava, Sanjeeva

2014-09-23

Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (p<0.05). All the significant spots were reproducible in all biological replicates and followed the same trend. Identification of the proteins from these spots revealed that nine proteins were common in both 2-DE and 2D-DIGE. These proteins are found to be involved in various cellular processes and pathways such as cytoskeleton function and cytokinesis, carbon, nitrogen, lipid metabolism, general translation and protein folding. Among these, our further study with the cytoskeletal proteins reveals that, compared to mitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin cytoskeleton and its regulators accumulated during meiosis by forming stable protein structure even though the corresponding mRNAs are degraded as cells enter into meiosis. This is in accordance with recent studies in higher eukaryotes where actin cytoskeleton is found to play vital role during meiotic chromosome segregation. Information generated by this study is significant to reveal that even though a cell that, unlike mitosis, is metabolically inactive with no isotropic bulging of membranes as buds (in meiosis) can require more actin cytoskeleton presumably to support nuclear movements. Copyright © 2014 Elsevier B.V. All rights reserved.

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

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

Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin

2014-03-28

Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined themore » impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.« less

Quantitative regulation of B cell division destiny by signal strength.

PubMed

Turner, Marian L; Hawkins, Edwin D; Hodgkin, Philip D

2008-07-01

Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

dbl-1/TGF-β and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest.

PubMed

Kaplan, Rebecca E W; Chen, Yutao; Moore, Brad T; Jordan, James M; Maxwell, Colin S; Schindler, Adam J; Baugh, L Ryan

2015-12-01

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

Aptamer Internalization via Endocytosis Inducing S-Phase Arrest and Priming Maver-1 Lymphoma Cells for Cytarabine Chemotherapy.

PubMed

Li, Huan; Yang, Shuanghui; Yu, Ge; Shen, Liangfang; Fan, Jia; Xu, Ling; Zhang, Hedong; Zhao, Nianxi; Zeng, Zihua; Hu, Tony; Wen, Jianguo; Zu, Youli

2017-01-01

The goal of precision therapy is to efficiently treat cancer without side effects. Aptamers are a class of small ligands composed of single-stranded oligonucleotides that bind to their targets with high affinity and specificity. In this study, we identified an ssDNA aptamer specifically targeting Maver-1 lymphoma cells with high binding affinity (K d = 70±8 pmol/L). Interestingly, cellular cycle studies revealed that exposure of Maver-1 cells to synthetic aptamers triggered S-phase arrest of 40% of the cells (vs. 18% baseline). Confocal microscopy confirmed specific cell binding of aptamers and the resultant endocytosis into Maver-1 cells. Subsequent functional assays validated the fact that aptamer internalization into targeted cells is a prerequisite for Maver-1 cell growth inhibition. Importantly, aptamer-induced S-phase arrest induced enhanced chemotherapeutic results involving cytarabine, which primarily kills lymphoma cells at S-phase. Combination treatments revealed that aptamer re-exposure considerably primed Maver-1 cells for cytarabine chemotherapy, thus achieving a synergistic killing effect by reaching cell death rates as high as 61% (vs. 13% or 14% induced by aptamer or cytarabine treatment alone). These findings demonstrated that aptamers do not only act as molecular ligands but can also function as biotherapeutic agents by inducing S-phase arrest of lymphoma cells. In addition, logical combination of aptamer and cytarabine treatments ushers the way to a unique approach in precision lymphoma chemotherapy.

Aptamer Internalization via Endocytosis Inducing S-Phase Arrest and Priming Maver-1 Lymphoma Cells for Cytarabine Chemotherapy

PubMed Central

Li, Huan; Yang, Shuanghui; Yu, Ge; Shen, Liangfang; Fan, Jia; Xu, Ling; Zhang, Hedong; Zhao, Nianxi; Zeng, Zihua; Hu, Tony; Wen, Jianguo; Zu, Youli

2017-01-01

The goal of precision therapy is to efficiently treat cancer without side effects. Aptamers are a class of small ligands composed of single-stranded oligonucleotides that bind to their targets with high affinity and specificity. In this study, we identified an ssDNA aptamer specifically targeting Maver-1 lymphoma cells with high binding affinity (Kd = 70±8 pmol/L). Interestingly, cellular cycle studies revealed that exposure of Maver-1 cells to synthetic aptamers triggered S-phase arrest of 40% of the cells (vs. 18% baseline). Confocal microscopy confirmed specific cell binding of aptamers and the resultant endocytosis into Maver-1 cells. Subsequent functional assays validated the fact that aptamer internalization into targeted cells is a prerequisite for Maver-1 cell growth inhibition. Importantly, aptamer-induced S-phase arrest induced enhanced chemotherapeutic results involving cytarabine, which primarily kills lymphoma cells at S-phase. Combination treatments revealed that aptamer re-exposure considerably primed Maver-1 cells for cytarabine chemotherapy, thus achieving a synergistic killing effect by reaching cell death rates as high as 61% (vs. 13% or 14% induced by aptamer or cytarabine treatment alone). These findings demonstrated that aptamers do not only act as molecular ligands but can also function as biotherapeutic agents by inducing S-phase arrest of lymphoma cells. In addition, logical combination of aptamer and cytarabine treatments ushers the way to a unique approach in precision lymphoma chemotherapy. PMID:28435459

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

PubMed

Song, Yi; Liu, Mei-Ju; Zhao, Guo-Wei; Qian, Jun-Jie; Dong, Yan; Liu, Hua; Sun, Guo-Jing; Mei, Zhu-Zhong; Liu, Bin; Tian, Bao-Lei; Sun, Zhi-Xian

2005-04-01

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

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

Tahara, Makiko; Department of Gastrointestinal Surgery, Jichi Medical University, Shimotsuke, Tochigi; Inoue, Takeshi

2013-05-17

Highlights: •Chemo-sensitivity to SN-38 was assayed by the automated cell counter. •Colon cancer cell line, HCT116 cells were more sensitive to SN-38 than HT29 cells. •Increase of cell size reflects G2/M arrest. •Appearance of small particles indicates cell apoptosis. -- Abstract: In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancermore » cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the {sup 3}H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.« less

Effects and possible mechanisms of simulated-microgravity on zebrafish embryonic cell

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Cellular level studies are helpful for revealing the underlying mechanisms of microgravity effects on living organisms. Many cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment. In this study, zebrafish embryonic cells (ZF4) were exposed to simulated-microgravity (SMG) for different times to investigate the effects and possible mechanisms of microgravity on fibroblasts. A significant arrest in G2/M phase was detected in ZF4 cells after 24 or 48 hour of SMG exposure, respectively. The mRNA levels of G2/M phase regulators cyclinB1 and cdc2 were significantly decreased, while wee1 was significantly increased. Additionally, CEP135, a core centrosome protein throughout the cell cycle, seems to play a key role in modulating this effect. Quantitative analysis showed that cep135 expression was significantly increased, while CEP135 protein expression level was significantly decreased two times after SMG. Further investigation demonstrated the transfection of dre-miR-22a, a miRNA for targeting cep135, also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells may due to the regulation of dre-miR-22a and its target cep135. Key Words: Simulated-microgravity; zebrafish embryonic cell; G2/M arrest; molecular mechanism

Polarized Cell Division of Chlamydia trachomatis

PubMed Central

Abdelrahman, Yasser; Ouellette, Scot P.; Belland, Robert J.; Cox, John V.

2016-01-01

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments. PMID:27505160

Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells.

PubMed

Morley, Karen L; Ferguson, Peter J; Koropatnick, James

2007-06-18

Tangeretin and nobiletin are citrus flavonoids that are among the most effective at inhibiting cancer cell growth in vitro and in vivo. The antiproliferative activity of tangeretin and nobiletin was investigated in human breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29. Both flavonoids inhibited proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at G1 in all three cell lines. At concentrations that resulted in significant inhibition of proliferation and cell cycle arrest, neither flavonoid induced apoptosis or cell death in any of the tumor cell lines. To test the ability of arrested cells to recover, cells that were incubated with tangeretin and nobiletin for 4 days were then cultured in flavonoid-free medium for an additional 4 days. Cells resumed proliferation similar to untreated control within a day of flavonoid removal. Cell cycle distribution was similar to that of control within 4 days of flavonoid removal. These data indicate that, in these cell lines at concentrations that inhibit proliferation up to 80% over 4 days, tangeretin and nobiletin are cytostatic and significantly suppress proliferation by cell cycle arrest without apoptosis. Such an agent could be expected to spare normal tissues from toxic side effects. Thus, tangeretin and nobiletin could be effective cytostatic anticancer agents. Inhibition of proliferation of human cancers without inducing cell death may be advantageous in treating tumors as it would restrict proliferation in a manner less likely to induce cytotoxicity and death in normal, non-tumor tissues.

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

Zhang, Chundong; Zhang, Ying; Li, Yi

Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent withmore » those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. - Highlights: • PRR11 started to increase in the late S phase and was retained until just before mitotic telophase. • PRR11-knockdown caused a significant cell cycle arrest in the late S phase and G2 phase. • The treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. • PRR11-knockdown led to multipolar spindles and multiple nuclei. • Forced expression of PRR11 promoted the PCC and inhibited cell proliferation.« less

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

PubMed

Bordonaro, M; Mariadason, J M; Aslam, F; Heerdt, B G; Augenlicht, L H

1999-10-01

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

NASA Technical Reports Server (NTRS)

Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

2000-01-01

Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

Mechanical stretch triggers rapid epithelial cell division through Piezo1.

PubMed

Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

2017-03-02

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

Synergistic effect of curcumin on epigallocatechin gallate-induced anticancer action in PC3 prostate cancer cells.

PubMed

Eom, Dae-Woon; Lee, Ji Hwan; Kim, Young-Joo; Hwang, Gwi Seo; Kim, Su-Nam; Kwak, Jin Ho; Cheon, Gab Jin; Kim, Ki Hyun; Jang, Hyuk-Jai; Ham, Jungyeob; Kang, Ki Sung; Yamabe, Noriko

2015-08-01

Epigallocatechin gallate (EGCG) and curcumin are well known to naturally-occurring anticancer agents. The aim of this study was to verify the combined beneficial anticancer effects of curcumin and EGCG on PC3 prostate cancer cells, which are resistant to chemotherapy drugs and apoptosis inducers. EGCG showed weaker inhibitory effect on PC3 cell proliferation than two other prostate cancer cell lines, LNCaP and DU145. Co-treatment of curcumin improved antiproliferative effect of EGCG on PC3 cells. The protein expressions of p21 were significantly increased by the co-treatment of EGCG and curcumin, whereas it was not changed by the treatment with each individual compound. Moreover, treatments of EGCG and curcumin arrested both S and G2/M phases of PC3 cells. These results suggest that the enhanced inhibitory effect of EGCG on PC3 cell proliferation by curcumin was mediated by the synergic up-regulation of p21-induced growth arrest and followed cell growth arrest.

Metabolism and the Control of Cell Fate Decisions and Stem Cell Renewal

PubMed Central

Ito, Kyoko; Ito, Keisuke

2016-01-01

Although the stem cells of various tissues remain in the quiescent state to maintain their undifferentiated state, they also undergo cell divisions as required, and if necessary, even a single stem cell is able to provide for lifelong tissue homeostasis. Stem cell populations are precisely controlled by the balance between their symmetric and asymmetric divisions, with their division patterns determined by whether the daughter cells involved retain their self-renewal capacities. Recent studies have reported that metabolic pathways and the distribution of mitochondria are regulators of the division balance of stem cells and that metabolic defects can shift division balance toward symmetric commitment, which leads to stem cell exhaustion. It has also been observed that in asymmetric division, old mitochondria, which are central metabolic organelles, are segregated to the daughter cell fated to cell differentiation, whereas in symmetric division, young and old mitochondria are equally distributed between both daughter cells. Thus, metabolism and mitochondrial biology play important roles in stem cell fate decisions. As these decisions directly affect tissue homeostasis, understanding their regulatory mechanisms in the context of cellular metabolism is critical. PMID:27482603

Metabolism and the Control of Cell Fate Decisions and Stem Cell Renewal.

PubMed

Ito, Kyoko; Ito, Keisuke

2016-10-06

Although the stem cells of various tissues remain in the quiescent state to maintain their undifferentiated state, they also undergo cell divisions as required, and if necessary, even a single stem cell is able to provide for lifelong tissue homeostasis. Stem cell populations are precisely controlled by the balance between their symmetric and asymmetric divisions, with their division patterns determined by whether the daughter cells involved retain their self-renewal capacities. Recent studies have reported that metabolic pathways and the distribution of mitochondria are regulators of the division balance of stem cells and that metabolic defects can shift division balance toward symmetric commitment, which leads to stem cell exhaustion. It has also been observed that in asymmetric division, old mitochondria, which are central metabolic organelles, are segregated to the daughter cell fated to cell differentiation, whereas in symmetric division, young and old mitochondria are equally distributed between both daughter cells. Thus, metabolism and mitochondrial biology play important roles in stem cell fate decisions. As these decisions directly affect tissue homeostasis, understanding their regulatory mechanisms in the context of cellular metabolism is critical.

Scorpion (Androctonus bicolor) venom exhibits cytotoxicity and induces cell cycle arrest and apoptosis in breast and colorectal cancer cell lines

PubMed Central

Al-Asmari, Abdulrahman K.; Riyasdeen, Anvarbatcha; Abbasmanthiri, Rajamohamed; Arshaduddin, Mohammed; Al-Harthi, Fahad Ali

2016-01-01

Objectives: The defective apoptosis is believed to play a major role in the survival and proliferation of neoplastic cells. Hence, the induction of apoptosis in cancer cells is one of the targets for cancer treatment. Researchers are considering scorpion venom as a potent natural source for cancer treatment because it contains many bioactive compounds. The main objective of the current study is to evaluate the anticancer property of Androctonus bicolor scorpion venom on cancer cells. Materials and Methods: Scorpions were milked by electrical stimulation of telsons and lyophilized. The breast (MDA-MB-231) and colorectal (HCT-8) cancer cells were maintained in appropriate condition. The venom cytotoxicity was assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the cellular and nuclear changes were studied with propidium iodide and 4’,6-diamidino-2-phenylindole stain, respectively. The cell cycle arrest was examined using muse cell analyzer. Results: The A. bicolor venom exerted cytotoxic effects on MDA-MB-231 and HCT-8 cells in a dose- and duration-dependent manner and induced apoptotic cell death. The treatment with this venom arrests the cancer cells in G0/G1 phase of cell cycle. Conclusions: The venom selectively induces the rate of apoptosis in MDA-MB-231 and HCT-8 cells as reflected by morphological and cell cycle studies. To the best of our knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest by A. bicolor scorpion venom. PMID:27721540

Scorpion (Androctonus bicolor) venom exhibits cytotoxicity and induces cell cycle arrest and apoptosis in breast and colorectal cancer cell lines.

PubMed

Al-Asmari, Abdulrahman K; Riyasdeen, Anvarbatcha; Abbasmanthiri, Rajamohamed; Arshaduddin, Mohammed; Al-Harthi, Fahad Ali

2016-01-01

The defective apoptosis is believed to play a major role in the survival and proliferation of neoplastic cells. Hence, the induction of apoptosis in cancer cells is one of the targets for cancer treatment. Researchers are considering scorpion venom as a potent natural source for cancer treatment because it contains many bioactive compounds. The main objective of the current study is to evaluate the anticancer property of Androctonus bicolor scorpion venom on cancer cells. Scorpions were milked by electrical stimulation of telsons and lyophilized. The breast (MDA-MB-231) and colorectal (HCT-8) cancer cells were maintained in appropriate condition. The venom cytotoxicity was assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the cellular and nuclear changes were studied with propidium iodide and 4',6-diamidino-2-phenylindole stain, respectively. The cell cycle arrest was examined using muse cell analyzer. The A. bicolor venom exerted cytotoxic effects on MDA-MB-231 and HCT-8 cells in a dose- and duration-dependent manner and induced apoptotic cell death. The treatment with this venom arrests the cancer cells in G0/G1 phase of cell cycle. The venom selectively induces the rate of apoptosis in MDA-MB-231 and HCT-8 cells as reflected by morphological and cell cycle studies. To the best of our knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest by A. bicolor scorpion venom.

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.

Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway

PubMed Central

ZHU, JIE; CHEN, MEIJUAN; CHEN, NING; MA, AIZHEN; ZHU, CHUNYAN; ZHAO, RUOLIN; JIANG, MIAO; ZHOU, JING; YE, LIHONG; FU, HAIAN; ZHANG, XU

2015-01-01

Glycyrrhetinic acid (GA) is a natural compound extracted from liquorice, which is often used in traditional Chinese medicine. The purpose of the present study was to investigate the antitumor effect of GA in human non-small cell lung cancer (NSCLC), and its underlying mechanisms in vitro. We have shown that GA suppressed the proliferation of A549 and NCI-H460 cells. Flow cytometric analysis showed that GA arrested cell cycle in G0/G1 phase without inducing apoptosis. Western blot analysis indicated that GA mediated G1-phase cell cycle arrest by upregulation of cyclin-dependent kinase inhibitors (CKIs) (p18, p16, p27 and p21) and inhibition of cyclins (cyclin-D1, -D3 and -E) and cyclin-dependent kinases (CDKs) (CDK4, 6 and 2). GA also maintained pRb phosphorylation status, and inhibited E2F transcription factor 1 (E2F-1) in both cell lines. GA upregulated the unfolded proteins, Bip, PERK and ERP72. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggered the unfolded protein response (UPR), which could be the mechanism by which GA inhibited cell proliferation in NSCLC cells. GA then coordinated the induction of ER chaperones, which decreased protein synthesis and induced cell cycle arrest in the G1 phase. This study provides experimental evidence to support the development of GA as a chemotherapeutic agent for NSCLC. PMID:25573651

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 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

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

Saldanha, Sabita N., E-mail: sabivan@uab.edu; Department of Biological Sciences, Alabama State University, Montgomery, AL 36104; Kala, Rishabh

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29more » colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells. - Highlights: • EGCG and NaB as a combination inhibits colorectal cancer cell proliferation. • The combination treatment induces DNA damage, G2/M and G1 arrest and apoptosis. • Survivin is effectively down-regulated by the combination treatment. • p21 and p53 expressions are induced by the combination treatment. • Epigenetic proteins DNMT1 and HDAC1 are effectively down-regulated by the treatment.« less

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest.

PubMed

Nguyen, Phong Dang; Gurevich, David Baruch; Sonntag, Carmen; Hersey, Lucy; Alaei, Sara; Nim, Hieu Tri; Siegel, Ashley; Hall, Thomas Edward; Rossello, Fernando Jaime; Boyd, Sarah Elizabeth; Polo, Jose Maria; Currie, Peter David

2017-07-06

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G 2 cell-cycle arrest within muscle stem cells, and disrupting this G 2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G 0 /G 1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells. Copyright © 2017. Published by Elsevier Inc.

COP9 Signalosome Subunit Csn8 Is Involved in Maintaining Proper Duration of the G1 Phase*

PubMed Central

Liu, Cheng; Guo, Li-Quan; Menon, Suchithra; Jin, Dan; Pick, Elah; Wang, Xuejun; Deng, Xing Wang; Wei, Ning

2013-01-01

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G1 phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G1 duration and affected expression of G1 regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence. PMID:23689509

CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

PubMed

Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

2016-11-16

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

Somatostatin Receptor-1 Induces Cell Cycle Arrest and Inhibits Tumor Growth in Pancreatic Cancer

PubMed Central

Li, Min; Wang, Xiaochi; Li, Wei; Li, Fei; Yang, Hui; Wang, Hao; Brunicardi, F. Charles; Chen, Changyi; Yao, Qizhi; Fisher, William E.

2010-01-01

Functional somatostatin receptors (SSTRs) are lost in human pancreatic cancer. Transfection of SSTR-1 inhibited pancreatic cancer cell proliferation in vitro. We hypothesize that stable transfection of SSTR-1 may inhibit pancreatic cancer growth in vivo possibly through cell cycle arrest. In this study, we examined the expression of SSTR-1 mRNA in human pancreatic cancer tissue specimens, and investigated the effect of SSTR-1 overexpression on cell proliferation, cell cycle, and tumor growth in in a subcutaneous nude mouse model. We found that SSTR-1 mRNA was downregulated in the majority of pancreatic cancer tissue specimens. Transfection of SSTR-1 caused cell cycle arrest at the G0/G1 growth phase, with a corresponding decline of cells in the S (mitotic) phase. The overexpression of SSTR-1 significantly inhibited subcutaneous tumor size by 71% and 43% (n=5, p<0.05, t-test), and inhibited tumor weight by 69% and 47%, (n=5, p<0.05, t-test), in Panc-SSTR-1 and MIA-SSTR-1 groups, respectively, indicating the potent inhibitory effect of SSTR-1 on pancreatic cancer growth. Our data demonstrate that overexpression of SSTR-1 significantly inhibits pancreatic cancer growth possibly through cell cycle arrest. This study suggests that gene therapy with SSTR-1 may be a potential adjuvant treatment for pancreatic cancer. PMID:18823376

Effect of primycin on growth-arrested cultures and cell integrity of Staphylococcus aureus.

PubMed

Feiszt, Péter; Schneider, György; Emődy, Levente

2017-06-01

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect.

A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

PubMed

Abd El-Hafeez, Amer Ali; Fujimura, Takashi; Kamei, Rikiya; Hirakawa, Noriko; Baba, Kenji; Ono, Kazuhisa; Kawamoto, Seiji

2017-07-14

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G 2 /M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21 Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G 2 /M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G 2 /M cell cycle arrest and apoptosis.

Asymmetric cell division of stem cells in the lung and other systems

PubMed Central

Berika, Mohamed; Elgayyar, Marwa E.; El-Hashash, Ahmed H. K.

2014-01-01

New insights have been added to identification, behavior and cellular properties of embryonic and tissue-specific stem cells over the last few years. The modes of stem cell division, asymmetric vs. symmetric, are tightly regulated during development and regeneration. The proper choice of a stem cell to divide asymmetrically or symmetrically has great consequences for development and disease because inappropriate asymmetric division disrupts organ morphogenesis, whereas uncontrolled symmetric division induces tumorigenesis. Therefore, understanding the behavior of lung stem cells could identify innovative solutions for restoring normal morphogenesis and/or regeneration of different organs. In this concise review, we describe recent studies in our laboratory about the mode of division of lung epithelial stem cells. We also compare asymmetric cell division (ACD) in the lung stem cells with other tissues in different organisms. PMID:25364740

Cell and plastid division are coordinated through the prereplication factor AtCDT1

PubMed Central

Raynaud, Cécile; Perennes, Claudette; Reuzeau, Christophe; Catrice, Olivier; Brown, Spencer; Bergounioux, Catherine

2005-01-01

The cell division cycle involves nuclear and cytoplasmic events, namely organelle multiplication and distribution between the daughter cells. Until now, plastid and plant cell division have been considered as independent processes because they can be uncoupled. Here, down-regulation of AtCDT1a and AtCDT1b, members of the prereplication complex, is shown to alter both nuclear DNA replication and plastid division in Arabidopsis thaliana. These data constitute molecular evidence for relationships between the cell-cycle and plastid division. Moreover, the severe developmental defects observed in AtCDT1-RNA interference (RNAi) plants underline the importance of coordinated cell and organelle division for plant growth and morphogenesis. PMID:15928083

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.

Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

PubMed

Sati, Leyla; Ovari, Laszlo; Bennett, David; Simon, Stephen D; Demir, Ramazan; Huszar, Gabor

2008-04-01

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

Long-range ordered vorticity patterns in living tissue induced by cell division

NASA Astrophysics Data System (ADS)

Rossen, Ninna S.; Tarp, Jens M.; Mathiesen, Joachim; Jensen, Mogens H.; Oddershede, Lene B.

2014-12-01

In healthy blood vessels with a laminar blood flow, the endothelial cell division rate is low, only sufficient to replace apoptotic cells. The division rate significantly increases during embryonic development and under halted or turbulent flow. Cells in barrier tissue are connected and their motility is highly correlated. Here we investigate the long-range dynamics induced by cell division in an endothelial monolayer under non-flow conditions, mimicking the conditions during vessel formation or around blood clots. Cell divisions induce long-range, well-ordered vortex patterns extending several cell diameters away from the division site, in spite of the system’s low Reynolds number. Our experimental results are reproduced by a hydrodynamic continuum model simulating division as a local pressure increase corresponding to a local tension decrease. Such long-range physical communication may be crucial for embryonic development and for healing tissue, for instance around blood clots.

The production of monokaryotic hyphae by Cryptococcus neoformans can be induced by high temperature arrest of the cell cycle and is independent of same-sex mating.

PubMed

Fu, Jianmin; Morris, Ian R; Wickes, Brian L

2013-01-01

Cryptococcus neoformans is a heterothallic fungal pathogen of humans and animals. Although the fungus grows primarily as a yeast, hyphae are produced during the sexual phase and during a process called monokaryotic fruiting, which is also believed to involve sexual reproduction, but between cells of the same mating type. Here we report a novel monokaryotic fruiting mechanism that is dependent on the cell cycle and occurs in haploid cells in the absence of sexual reproduction. Cells grown at 37°C were found to rapidly produce hyphae (∼4 hrs) and at high frequency (∼40% of the population) after inoculation onto hyphae-inducing agar. Microscopic examination of the 37°C seed culture revealed a mixture of normal-sized and enlarged cells. Micromanipulation of single cells demonstrated that only enlarged cells were able to produce hyphae and genetic analysis confirmed that hyphae did not arise from α-α mating or endoduplication. Cell cycle analysis revealed that cells grown at 37°C had an increased population of cells in G2 arrest, with the proportion correlated with the frequency of monokaryotic fruiting. Cell sorting experiments demonstrated that enlarged cells were only found in the G2-arrested population and only this population contained cells able to produce hyphae. Treatment of cells at low temperature with the G2 cell cycle arrest agent, nocodazole, induced hyphal growth, confirming the role of the cell cycle in this process. Taken together, these results reveal a mating-independent mechanism for monokaryotic fruiting, which is dependent on the cell cycle for induction of hyphal competency.

Hydroquinone induces TK6 cell growth arrest and apoptosis through PARP-1/p53 regulatory pathway.

PubMed

Luo, Hao; Liang, Hairong; Chen, Jiajia; Xu, Yongchun; Chen, Yuting; Xu, Longmei; Yun, Lin; Liu, Jiaxian; Yang, Hui; Liu, Linhua; Peng, Jianming; Liu, Zhidong; Tang, Lin; Chen, Wen; Tang, Huanwen

2017-09-01

Hydroquinone (HQ), one of the most important metabolites derived from benzene, induces cell cycle arrest and apoptosis. Poly(ADP-ribose) polymerase-1 (PARP-1) participates in various biological processes, including DNA repair and cell cycle regulation. To explore whether PARP-1 regulatory pathway mediated HQ-induced cell cycle arrest and apoptosis, we assessed the effect of PARP-1 suppression on induction of apoptosis analyzed by FACSCalibur flow cytometer in PARP-1 deficientTK6 cells (TK6-shPARP-1). We observed an increase in the fraction of cells in G1 phase by 7.6% and increased apoptosis by 4.5% in PARP-1-deficient TK6 cells (TK6-shPARP-1) compared to those negative control cells (TK6-shNC cells) in response to HQ treatment. Furthermore, HQ might activate the extrinsic pathways of apoptosis via up-regulation of Fas expression, followed by caspase-3 activation, apoptotic body, and sub G1 accumulation. Enhanced p53 expression was observed in TK6-shPARP-1 cells than in TK6-shNC cells after HQ treatment. In contrast, Fas expression was lower in TK6-shPARP-1 cells than in TK6-shNC cells. Therefore, we conclude that HQ may activate apoptotic signals via Fas up-regulation and p53-mediated apoptosis in TK6-shNC cells. The reduction of PARP-1 expression further intensified up-regulation of p53 in TK6-shPARP-1 cells, resulting in an increased G1→S phase cell arrest and apoptosis in TK6-shPARP-1 cells compared to TK6-shNC cells. © 2017 Wiley Periodicals, Inc.

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 on g2/m phase cell cycle distribution and aneuploidy formation of exposure to a 60 Hz electromagnetic field in combination with ionizing radiation or hydrogen peroxide in l132 nontumorigenic human lung epithelial cells.

PubMed

Jin, Hee; Yoon, Hye Eun; Lee, Jae-Seon; Kim, Jae-Kyung; Myung, Sung Ho; Lee, Yun-Sil

2015-03-01

The aim of the present study was to assess whether exposure to the combination of an extremely low frequency magnetic field (ELF-MF; 60 Hz, 1 mT or 2 mT) with a stress factor, such as ionizing radiation (IR) or H2O2, results in genomic instability in non-tumorigenic human lung epithelial L132 cells. To this end, the percentages of G2/M-arrested cells and aneuploid cells were examined. Exposure to 0.5 Gy IR or 0.05 mM H2O2 for 9 h resulted in the highest levels of aneuploidy; however, no cells were observed in the subG1 phase, which indicated the absence of apoptotic cell death. Exposure to an ELF-MF alone (1 mT or 2 mT) did not affect the percentages of G2/M-arrested cells, aneuploid cells, or the populations of cells in the subG1 phase. Moreover, when cells were exposed to a 1 mT or 2 mT ELF-MF in combination with IR (0.5 Gy) or H2O2 (0.05 mM), the ELF-MF did not further increase the percentages of G2/M-arrested cells or aneuploid cells. These results suggest that ELF-MFs alone do not induce either G2/M arrest or aneuploidy, even when administered in combination with different stressors.

Androgen Control of Cell Proliferation and Cytoskeletal Reorganization in Human Fibrosarcoma Cells

PubMed Central

Chauhan, Sanjay; Kunz, Susan; Davis, Kelli; Roberts, Jordan; Martin, Greg; Demetriou, Manolis C.; Sroka, Thomas C.; Cress, Anne E.; Miesfeld, Roger L.

2009-01-01

We recently generated an HT-1080-derived cell line called HT-AR1 that responds to dihydrotestosterone (DHT) treatment by undergoing cell growth arrest in association with cytoskeletal reorganization and induction of neuroendocrine-like cell differentiation. In this report, we show that DHT induces a dose-dependent increase in G0/G1 growth-arrested cells using physiological levels of hormone. The arrested cells increase in cell size and contain a dramatic redistribution of desmoplakin, keratin 5, and chromogranin A proteins. DHT-induced cytoskeletal changes were also apparent from time lapse video microscopy that showed that androgen treatment resulted in the rapid appearance of neuronal-like membrane extensions. Expression profiling analysis using RNA isolated from DHT-treated HT-AR1 cells revealed that androgen receptor activation leads to the coordinate expression of numerous cell signaling genes including RhoB, PTGF-β, caveolin-2, Egr-1, myosin 1B, and EHM2. Because RhoB has been shown to have a role in tumor suppression and neuronal differentiation in other cell types, we investigated RhoB signaling functions in the HT-AR1 steroid response. We found that steroid induction of RhoB was DHT-specific and that newly synthesized RhoB protein was post-translationally modified and localized to endocytic vesicles. Moreover, treatment with a farnesyl transferase inhibitor reduced DHT-dependent growth arrest, suggesting that prenylated RhoB might function to inhibit HT-AR1 cell proliferation. This was directly shown by transfecting HT-AR1 cells with RhoB coding sequences containing activating or dominant negative mutations. PMID:14576147

Targeted Approaches to Overcoming Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2011-08-01

NM_001012271 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog AF053305 CDC20 Cell division cycle 20 homolog BG256659 CDC25B Cell division cycle...by benzimidazoles 1 homolog), BIRC5/ Survivin, CDCA8 (cell division cycle-associated protein 8), AURKB (aurora kinase B), CDC25B (cell division cycle

Nanoporous capsules of block co-polymers of [(MeO-PEG-NH)-b-(L-GluA)]-PCL for the controlled release of anticancer drugs for therapeutic applications.

PubMed

Amgoth, Chander; Dharmapuri, Gangappa; Kalle, Arunasree M; Paik, Pradip

2016-03-29

Herein, new nanoporous capsules of the block co-polymers of MeO-PEG-NH-(L-GluA)10 and polycaprolactone (PCL) have been synthesized through a surfactant-free cost-effective self-assembled soft-templating approach for the controlled release of drugs and for therapeutic applications. The nanoporous polymer capsules are designed to be biocompatible and are capable of encapsulating anticancer drugs (e.g., doxorubicin hydrochloride (DOX) and imatinib mesylate (ITM)) with a high extent (∼279 and ∼480 ng μg(-1), respectively). We have developed a nanoformulation of porous MeO-PEG-NH-(L-GluA)10-PCL capsules with DOX and ITM. The porous polymer nanoformulations have been programmed in terms of the release of anticancer drugs with a desired dose to treat the leukemia (K562) and human carcinoma cells (HepG2) in vitro and show promising IC50 values with a very high mortality of cancer cells (up to ∼96.6%). Our nanoformulation arrests the cell divisions due to 'cellular scenescence' and kills the cancer cells specifically. The present findings could enrich the effectiveness of idiosyncratic nanoporous polymer capsules for use in various other nanomedicinal and biomedical applications, such as for killing cancer cells, immune therapy, and gene delivery.

Nanoporous capsules of block co-polymers of [(MeO-PEG-NH)-b-(L-GluA)]-PCL for the controlled release of anticancer drugs for therapeutic applications

NASA Astrophysics Data System (ADS)

Amgoth, Chander; Dharmapuri, Gangappa; Kalle, Arunasree M.; Paik, Pradip

2016-03-01

Herein, new nanoporous capsules of the block co-polymers of MeO-PEG-NH-(L-GluA)10 and polycaprolactone (PCL) have been synthesized through a surfactant-free cost-effective self-assembled soft-templating approach for the controlled release of drugs and for therapeutic applications. The nanoporous polymer capsules are designed to be biocompatible and are capable of encapsulating anticancer drugs (e.g., doxorubicin hydrochloride (DOX) and imatinib mesylate (ITM)) with a high extent (˜279 and ˜480 ng μg-1, respectively). We have developed a nanoformulation of porous MeO-PEG-NH-(L-GluA)10-PCL capsules with DOX and ITM. The porous polymer nanoformulations have been programmed in terms of the release of anticancer drugs with a desired dose to treat the leukemia (K562) and human carcinoma cells (HepG2) in vitro and show promising IC50 values with a very high mortality of cancer cells (up to ˜96.6%). Our nanoformulation arrests the cell divisions due to ‘cellular scenescence’ and kills the cancer cells specifically. The present findings could enrich the effectiveness of idiosyncratic nanoporous polymer capsules for use in various other nanomedicinal and biomedical applications, such as for killing cancer cells, immune therapy, and gene delivery.

Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

PubMed

Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

2015-06-01

Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. © 2015 American Society of Plant Biologists. All Rights Reserved.

ATM and MET kinases are synthetic lethal with non-genotoxic activation of p53

PubMed Central

Sullivan, Kelly D.; Padilla-Just, Nuria; Henry, Ryan E.; Porter, Christopher C.; Kim, Jihye; Tentler, John J.; Eckhardt, S. Gail; Tan, Aik Choon; DeGregori, James; Espinosa, Joaquín M.

2012-01-01

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a ‘Synthetic Lethal with Nutlin-3’ genome-wide shRNA screen, which revealed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors enable Nutlin-3 to kill tumor spheroids. These results identify novel pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies. PMID:22660439

p21 induction plays a dual role in anti-cancer activity of ursolic acid

PubMed Central

Zhang, Xudong; Song, Xinhua; Yin, Shutao; Zhao, Chong; Fan, Lihong

2015-01-01

Previous studies have shown that induction of G1 arrest and apoptosis by ursolic acid is associated with up-regulation of cyclin-dependent kinase inhibitor (CDKI) protein p21 in multiple types of cancer cells. However, the functional role of p21 induction in G1 cell cycle arrest and apoptosis, and the mechanisms of p21 induction by ursolic acid have not been critically addressed. In the current study, we demonstrated that p21 played a mediator role in G1 cell cycle arrest by ursolic acid, whereas p21-mediated up-regulation of Mcl-1 compromised apoptotic effect of ursolic acid. These results suggest that p21 induction plays a dual role in the anti-cancer activity of ursolic acid in terms of cell cycle and apoptosis regulation. p21 induction by ursolic acid was attributed to p53 transcriptional activation. Moreover, we found that ursolic acid was able to inhibit murine double minute-2 protein (MDM2) and T-LAK cell-originated protein kinase (TOPK), the two negative regulator of p53, which in turn contributed to ursolic acid-induced p53 activation. Our findings provided novel insights into understanding of the mechanisms involved in cell cycle arrest and apoptosis induction in response to ursolic acid exposure. PMID:26582056

Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin-Induced Cell Cycle Arrest and Apoptosis in Cancer Cells.

PubMed

Shrestha, Anup; Nepal, Saroj; Kim, Mi Jin; Chang, Jae Hoon; Kim, Sang-Hyun; Jeong, Gil-Saeng; Jeong, Chul-Ho; Park, Gyu Hwan; Jung, Sunghee; Lim, Jaecheong; Cho, Eunha; Lee, Soyoung; Park, Pil-Hoon

2016-02-01

Adiponectin predominantly secreted from adipose tissue has exhibited potent anti-proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti-cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF-7) cancer cells. Gene ablation of FoxO3A prevented gAcrp-induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF-7 cells. Transfection with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp-induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of FoxO3A in HepG2 and MCF-7 cells. In addition, suppression of AMPK also blocked gAcrp-induced cell cycle arrest and further attenuated gAcrp-induced caspase-3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp-induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti-proliferative effect of adiponectin in cancer cells. © 2015 Wiley Periodicals, Inc.

Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy.

PubMed

Suzuki, Masatoshi; Boothman, David A

2008-03-01

Replicative senescence is a fundamental feature in normal human diploid cells and results from dysfunctional telomeres at the Hayflick cell division limit. Ionizing radiation (IR) prematurely induces the same phenotypes as replicative senescence prior to the Hayflick limit. This process is known as stress-induced premature senescence (SIPS). Since the cell cycle is irreversibly arrested in SIPS-induced cells, even if they are stimulated by various growth factors, it is thought that SIPS is a form of cell death, irreversibly eliminating replicating cells. IR-induced-focus formation of DNA repair proteins, a marker of DNA damage, is detected in SIPS as well as replicative senescent cells. Furthermore, both processes persistently induce cell cycle checkpoint mechanisms, indicating DNA damage created by ionizing radiation induces SIPS in normal cells, possibly by the same mechanisms as those occurring in replicative senescence. Interestingly, IR induces SIPS not only in normal cells, but also in tumor cells. Due to the expression of telomerase in tumor cells, telomere-dependent replicative senescence does not occur. However, SIPS is induced under certain conditions after IR exposure. Thus, cell death triggered by IR can be attributed to apoptosis or SIPS in tumor cells. However, metabolic function remains intact in SIPS-induced cancer cells, and recent studies show that senescence eliminate cells undergoing SIPS secrete various kinds of factors outside the cell, changing the microenvironment. Evidence using co-culture systems containing normal senescent stromal cells and epithelial tumor cells show that factors secreted from senescent stroma cells promote the growth of tumor epithelial cells both in vitro and in vivo. Thus, regulation of factors secreted from SIPS-induced stromal cells, as well as tumor cells, may affect radiotherapy.

Effect of caffeine on radiation-induced mitotic delay: delayed expression of G/sub 2/ arrest

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

Rowley, R.; Zorch, M.; Leeper, D.B.

1984-01-01

In the presence of 5 mM caffeine, irradiated (1.5 Gy) S and G/sub 2/ cells progressed to mitosis in register and without arrest in G/sub 2/. Caffeine (5 mM) markedly reduced mitotic delay even after radiation doses up to 20 Gy. When caffeine was removed from irradiated (1.5 Gy) and caffeine-treated cells, a period of G/sub 2/ arrest followed, similar in length to that produced by radiation alone. The arrest expressed was independent of the duration of the caffeine treatment for exposures up to 3 hr. The similarity of the response to the cited effects of caffeine on S-phase delaymore » suggests a common basis for delay induction in S and G/sub 2/ phases.« less

dbl-1/TGF-β and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest

PubMed Central

Moore, Brad T.; Jordan, James M.; Maxwell, Colin S.; Schindler, Adam J.; Baugh, L. Ryan

2015-01-01

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This “L1 arrest” (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development. PMID:26656736

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

PubMed

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations †

PubMed Central

Rivkin, Richard B.

1986-01-01

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With 68Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with 68Ge(OH)4 and NaH14CO3, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom. PMID:16347039

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

NASA Astrophysics Data System (ADS)

Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

2006-06-01

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

PubMed Central

Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

2016-01-01

Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

Division Planes Alternate in Spherical Cells of Escherichia coli

PubMed Central

Begg, K. J.; Donachie, W. D.

1998-01-01

In the spherical cells of Escherichia coli rodA mutants, division is initiated at a single point, from which a furrow extends progressively around the cell. Using “giant” rodA ftsA cells, we confirmed that each new division furrow is initiated at the midpoint of the previous division plane and runs perpendicular to it. PMID:9573213

The final cut: cell polarity meets cytokinesis at the bud neck in S. cerevisiae.

PubMed

Juanes, Maria Angeles; Piatti, Simonetta

2016-08-01

Cell division is a fundamental but complex process that gives rise to two daughter cells. It includes an ordered set of events, altogether called "the cell cycle", that culminate with cytokinesis, the final stage of mitosis leading to the physical separation of the two daughter cells. Symmetric cell division equally partitions cellular components between the two daughter cells, which are therefore identical to one another and often share the same fate. In many cases, however, cell division is asymmetrical and generates two daughter cells that differ in specific protein inheritance, cell size, or developmental potential. The budding yeast Saccharomyces cerevisiae has proven to be an excellent system to investigate the molecular mechanisms governing asymmetric cell division and cytokinesis. Budding yeast is highly polarized during the cell cycle and divides asymmetrically, producing two cells with distinct sizes and fates. Many components of the machinery establishing cell polarization during budding are relocalized to the division site (i.e., the bud neck) for cytokinesis. In this review we recapitulate how budding yeast cells undergo polarized processes at the bud neck for cell division.

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

PubMed

Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

2017-11-22

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

[Efficacy of HSV-tk/GCV system on human laryngeal squamous cell cancer in vitro].

PubMed

Ding, Xiu-yong; Qin, Yong; Li, Fu-ying; Cong, Tie-chuan

2006-05-01

Efficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed. Hep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay. HSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred. The HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Curcumin Induces G2/M Arrest and Apoptosis in Cisplatin-Resistant Human Ovarian Cancer Cells by Modulating Akt and p38 MAPK

PubMed Central

Weir, Nathan M.; Selvendiran, Karuppaiyah; Kutala, Vijay Kumar; Tong, Liyue; Vishwanath, Shilpa; Rajaram, Murugesan; Tridandapani, Susheela; Anant, Shrikant; Kuppusamy, Periannan

2007-01-01

Curcumin, a major active component of turmeric, is known to induce apoptosis in several types of cancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the present study was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovarian cancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin-resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxide generation was observed in both CR and CS cells treated with curcumin. Curcumin induced G2/M phase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through the activation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylation of Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed that curcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the induction of superoxide generation, G2/M arrest, and apoptosis. PMID:17218783

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.

Mechanisms of Regulating Tissue Elongation in Drosophila Wing: Impact of Oriented Cell Divisions, Oriented Mechanical Forces, and Reduced Cell Size

PubMed Central

Li, Yingzi; Naveed, Hammad; Kachalo, Sema; Xu, Lisa X.; Liang, Jie

2014-01-01

Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. Here we use a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. We simulate the effects of directional cues on tissue elongation. We also computationally analyze the role of reduced cell size. Our simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. We show that oriented cell divisions and oriented mechanical forces act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, we raise the novel hypothesis that reduced cell size may significantly promote tissue elongation. We find that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, our simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Our simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation. PMID:24504016

Achieving Precision Death with Cell-Cycle Inhibitors that Target DNA Replication and Repair.

PubMed

Lin, Aimee Bence; McNeely, Samuel C; Beckmann, Richard P

2017-07-01

All cancers are characterized by defects in the systems that ensure strict control of the cell cycle in normal tissues. The consequent excess tissue growth can be countered by drugs that halt cell division, and, indeed, the majority of chemotherapeutics developed during the last century work by disrupting processes essential for the cell cycle, particularly DNA synthesis, DNA replication, and chromatid segregation. In certain contexts, the efficacy of these classes of drugs can be impressive, but because they indiscriminately block the cell cycle of all actively dividing cells, their side effects severely constrain the dose and duration with which they can be administered, allowing both normal and malignant cells to escape complete growth arrest. Recent progress in understanding how cancers lose control of the cell cycle, coupled with comprehensive genomic profiling of human tumor biopsies, has shown that many cancers have mutations affecting various regulators and checkpoints that impinge on the core cell-cycle machinery. These defects introduce unique vulnerabilities that can be exploited by a next generation of drugs that promise improved therapeutic windows in patients whose tumors bear particular genomic aberrations, permitting increased dose intensity and efficacy. These developments, coupled with the success of new drugs targeting cell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newer strategies that may facilitate better therapeutic targeting of drugs that inhibit the various components that safeguard the fidelity of the fundamental processes of DNA replication and repair. Clin Cancer Res; 23(13); 3232-40. ©2017 AACR . ©2017 American Association for Cancer Research.

Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells.

PubMed

Basu, Gargi D; Pathangey, Latha B; Tinder, Teresa L; Gendler, Sandra J; Mukherjee, Pinku

2005-01-01

Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines. MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model. The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G0/G1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and increased necrosis in the tumor mass. The disparate molecular mechanisms of celecoxib-induced growth inhibition in human breast cancer cells depends upon the level of COX-2 expression and the invasive potential of the cell lines examined. Data suggest a role for COX-2 not only in the growth of cancer cells but also in activating the angiogenic pathway through regulating levels of vascular endothelial growth factor.

Increased expression of cyclin B1 mRNA coincides with diminished G{sub 2}-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

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

Bernhard, E.J.; Maity, A.; McKenna, W.G.

1994-12-01

The irradiation of cells results in delayed progression through the G{sub 2} phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G{sub 2}-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G{sub 2}-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G{sub 2}-phase arrest. In HeLamore » cells, the G{sub 2}-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G{sub 2}-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G{sub 2}-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs.« less

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

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

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Lossmore » of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.« less

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

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

Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.

2008-07-15

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cellsmore » treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.« less

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

PubMed

Kapoor, Vaishali; Aggarwal, Sadhna; Das, Satya N

2016-04-01

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells. Copyright © 2016 John Wiley & Sons, Ltd.

Quantifying cell turnover using CFSE data.

PubMed

Ganusov, Vitaly V; Pilyugin, Sergei S; de Boer, Rob J; Murali-Krishna, Kaja; Ahmed, Rafi; Antia, Rustom

2005-03-01

The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based "Smith-Martin" model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S+G2+M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett-Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle.

β2-adrenoceptor blockage induces G1/S phase arrest and apoptosis in pancreatic cancer cells via Ras/Akt/NFκB pathway.

PubMed

Zhang, Dong; Ma, Qingyong; Wang, Zheng; Zhang, Min; Guo, Kun; Wang, Fengfei; Wu, Erxi

2011-11-26

Smoking and stress, pancreatic cancer (PanCa) risk factors, stimulate nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and catecholamines production respectively. NNK and catecholamine bind the β-adrenoceptors and induce PanCa cell proliferation; and we have previously suggested that β-adrenergic antagonists may suppress proliferation and invasion and stimulate apoptosis in PanCa. To clarify the mechanism of apoptosis induced by β2-adrenergic antagonist, we hypothesize that blockage of the β2-adrenoceptor could induce G1/S phase arrest and apoptosis and Ras may be a key player in PanCa cells. The β1 and β2-adrenoceptor proteins were detected on the cell surface of PanCa cells from pancreatic carcinoma specimen samples by immunohistochemistry. The β2-adrenergic antagonist ICI118,551 significantly induced G1/S phase arrest and apoptosis compared with the β1-adrenergic antagonist metoprolol, which was determined by the flow cytometry assay. β2-adrenergic antagonist therapy significantly suppressed the expression of extracellular signal-regulated kinase, Akt, Bcl-2, cyclin D1, and cyclin E and induced the activation of caspase-3, caspase-9 and Bax by Western blotting. Additionally, the β2-adrenergic antagonist reduced the activation of NFκB in vitro cultured PanCa cells. The blockage of β2-adrenoceptor markedly induced PanCa cells to arrest at G1/S phase and consequently resulted in cell death, which is possibly due to that the blockage of β2-adrenoceptor inhibited NFκB, extracellular signal-regulated kinase, and Akt pathways. Therefore, their upstream molecule Ras may be a key factor in the β2-adrenoceptor antagonist induced G1/S phase arrest and apoptosis in PanCa cells. The new pathway discovered in this study may provide an effective therapeutic strategy for PanCa.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.

PubMed

Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio

2010-11-01

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G₂ arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest. Additionally our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression

PubMed Central

Soo, Julia K; MacKenzie Ross, Alastair D; Kallenberg, David M; Milagre, Carla; Heung Chong, W; Chow, Jade; Hill, Lucy; Hoare, Stacey; Collinson, Rebecca S; Hossain, Mehnaz; Keith, W Nicol; Marais, Richard; Bennett, Dorothy C

2011-01-01

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: β-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event. PMID:21418545

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.

Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: Lessons from the human hair follicle matrix.

PubMed

Purba, Talveen S; Brunken, Lars; Peake, Michael; Shahmalak, Asim; Chaves, Asuncion; Poblet, Enrique; Ceballos, Laura; Gandarillas, Alberto; Paus, Ralf

2017-09-01

Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21 CIP1 , p27 KIP1 and p57 KIP2 ) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21 CIP1 , p27 KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57 KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

PubMed

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

2014-04-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

PubMed Central

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

2014-01-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest

PubMed Central

Kalayda, Ganna V.; Mannewitz, Mareike; Cinatl, Jindrich; Rothweiler, Florian; Michaelis, Martin; Saafan, Hisham; Ritter, Christoph A.; Jaehde, Ulrich

2017-01-01

The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis. PMID:28746345

A single-cell pedigree analysis of alternative stochastic lymphocyte fates

PubMed Central

Hawkins, E. D.; Markham, J. F.; McGuinness, L. P.; Hodgkin, P. D.

2009-01-01

In contrast to most stimulated lymphocytes, B cells exposed to Toll-like receptor 9 ligands are nonself-adherent, allowing individual cells and families to be followed in vitro for up to 5 days. These B cells undergo phases typical of an adaptive response, dividing up to 6 times before losing the impetus for further growth and division and eventually dying by apoptosis. Using long-term microscopic imaging, accurate histories of individual lymphocyte fates were collected. Quantitative analysis of family relationships revealed that times to divide of siblings were strongly related but these correlations were progressively lost through consecutive divisions. A weaker, but significant, correlation was also found for death times among siblings. Division cessation is characterized by a loss of cell growth and the division in which this occurs is strongly inherited from the original founder cell and is related to the size this cell reaches before its first division. Thus, simple division-based dilution of factors synthesized during the first division may control the maximum division reached by stimulated cells. The stochastic distributions of times to divide, times to die, and divisions reached are also measured. Together, these results highlight the internal cellular mechanisms that control immune responses and provide a foundation for the development of new mathematical models that are correct at both single-cell and population levels. PMID:19633185

Cell Division Synchronization

DTIC Science & Technology

The report summarizes the progress in the design and construction of automatic equipment for synchronizing cell division in culture by periodic...Concurrent experiments in hypothermic synchronization of algal cell division are reported.

Gravity and the orientation of cell division

NASA Technical Reports Server (NTRS)

Helmstetter, C. E.

1997-01-01

A novel culture system for mammalian cells was used to investigate division orientations in populations of Chinese hamster ovary cells and the influence of gravity on the positioning of division axes. The cells were tethered to adhesive sites, smaller in diameter than a newborn cell, distributed over a nonadhesive substrate positioned vertically. The cells grew and divided while attached to the sites, and the angles and directions of elongation during anaphase, projected in the vertical plane, were found to be random with respect to gravity. However, consecutive divisions of individual cells were generally along the same axis or at 90 degrees to the previous division, with equal probability. Thus, successive divisions were restricted to orthogonal planes, but the choice of plane appeared to be random, unlike the ordered sequence of cleavage orientations seen during early embryo development.

Exploring a Link Between NF-KB and G2/M Cell Cycle Arrest in Breast Cancer Cells

DTIC Science & Technology

2005-04-01

studies with esophageal squamous cell carcinom a lines have shown that IR induced p21waf1/ ciP ’ and a G2 cell cycle arrest that could als o be...i AD Award Number : DAMD17-02-1-062 3 TITLE : Exploring a Link Between NF-KB and G 2 /M Cell Cycle Arres t in Breast Cancer Cell s PRINCIPAL...Mar 2005 ) 4 . TITLE AND SUBTITL E Exploring a Link Between NF-kB and G 2 /M Cell Cycle Arres t in Breast Cancer Cells 5. FUND/NG NUMBERS DAMD17-02-1

CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization.

PubMed

Mandakovic, Dinka; Trigo, Carla; Andrade, Derly; Riquelme, Brenda; Gómez-Lillo, Gabriela; Soto-Liebe, Katia; Díez, Beatriz; Vásquez, Mónica

2016-01-01

Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120.

Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells

PubMed Central

Burke, Russell T.; Marcus, Joshua M.; Orth, James D.

2017-01-01

Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers. PMID:28467801

Nature of the Nucleosomal Barrier to RNA Polymerase II | Center for Cancer Research

Cancer.gov

In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic inactivation (arrest) of the pol II core enzyme. To determine potential mechanisms making nucleosomes transparent to transcription in vivo, we analyzed the nature of the nucleosome-induced arrest. We found that the arrests

The stem cell division theory of cancer.

PubMed

López-Lázaro, Miguel

2018-03-01

All cancer registries constantly show striking differences in cancer incidence by age and among tissues. For example, lung cancer is diagnosed hundreds of times more often at age 70 than at age 20, and lung cancer in nonsmokers occurs thousands of times more frequently than heart cancer in smokers. An analysis of these differences using basic concepts in cell biology indicates that cancer is the end-result of the accumulation of cell divisions in stem cells. In other words, the main determinant of carcinogenesis is the number of cell divisions that the DNA of a stem cell has accumulated in any type of cell from the zygote. Cell division, process by which a cell copies and separates its cellular components to finally split into two cells, is necessary to produce the large number of cells required for living. However, cell division can lead to a variety of cancer-promoting errors, such as mutations and epigenetic mistakes occurring during DNA replication, chromosome aberrations arising during mitosis, errors in the distribution of cell-fate determinants between the daughter cells, and failures to restore physical interactions with other tissue components. Some of these errors are spontaneous, others are promoted by endogenous DNA damage occurring during quiescence, and others are influenced by pathological and environmental factors. The cell divisions required for carcinogenesis are primarily caused by multiple local and systemic physiological signals rather than by errors in the DNA of the cells. As carcinogenesis progresses, the accumulation of DNA errors promotes cell division and eventually triggers cell division under permissive extracellular environments. The accumulation of cell divisions in stem cells drives not only the accumulation of the DNA alterations required for carcinogenesis, but also the formation and growth of the abnormal cell populations that characterize the disease. This model of carcinogenesis provides a new framework for understanding the disease and has important implications for cancer prevention and therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Neuronal injury from cardiac arrest: aging years in minutes.

PubMed

Cherry, Brandon H; Sumien, Nathalie; Mallet, Robert T

2014-01-01

Cardiac arrest is a leading cause of death and permanent disability. Most victims succumb to the oxidative and inflammatory damage sustained during cardiac arrest/resuscitation, but even survivors typically battle long-term neurocognitive impairment. Although extensive research has delineated the complex mechanisms that culminate in neuronal damage and death, no effective treatments have been developed to interrupt these mechanisms. Of importance, many of these injury cascades are also active in the aging brain, where neurons and other cells are under persistent oxidative and inflammatory stress which eventually damages or kills the cells. In light of these similarities, it is reasonable to propose that the brain essentially ages the equivalent of several years within the few minutes taken to resuscitate a patient from cardiac arrest. Accordingly, cardiac arrest-resuscitation models may afford an opportunity to study the deleterious mechanisms underlying the aging process, on an accelerated time course. The aging and resuscitation fields both stand to gain pivotal insights from one another regarding the mechanisms of injury sustained during resuscitation from cardiac arrest and during aging. This synergism between the two fields could be harnessed to foster development of treatments to not only save lives but also to enhance the quality of life for the elderly.

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

PubMed Central

Wang, Lintao; Peng, Yanyan; Shi, Kaikai; Wang, Haixiao; Lu, Jianlei; Li, Yanli; Ma, Changyan

2015-01-01

Abstract Recent studies have revealed that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, a traditional Chinese medicine, possesses anticancer activity. However, its effect on breast cancer cells so far has not been elucidated clearly. In the present study, we evaluated the effects of osthole on the proliferation, cell cycle and apoptosis of human breast cancer cells MDA-MB 435. We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells, The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole, as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation. The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression. Were observed taken together, these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer. PMID:25859268

Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro.

PubMed

Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

2013-08-06

Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma.

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in Candida albicans.

PubMed

Yun, JiEun; Lee, Dong Gun

2017-03-01

Chlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in Candida albicans. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels. To evaluate the inhibitory effects on CRA-induced cell death, C. albicans cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL. CRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in C. albicans. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked. CRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in C. albicans. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes. This study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis. Copyright © 2016. Published by Elsevier B.V.

Cytotoxicity of flavones and flavonols to a human esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G2/M arrest and apoptosis.

PubMed

Zhang, Qiang; Zhao, Xin-Huai; Wang, Zhu-Jun

2009-08-01

In this study, cytotoxic effects of structurally related flavones and flavonols on a human esophageal squamous cell carcinoma cell line (KYSE-510) were determined, and the molecular mechanisms responsible for their cytotoxic effects were studied. The results of MTT assay showed that flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) were able to induce cytotoxicity in KYSE-510 cells in a dose- and time-dependent manner, and the cytotoxic potency of these compounds was in the order of: luteolin>quercetin>chrysin>kaempferol>apigenin>myricetin. Flow cytometry and DNA fragmentation analysis indicated that the cytotoxicity induced by flavones and flavonols was mediated by G(2)/M cell cycle arrest and apoptosis. Furthermore, the expression of genes related to cell cycle arrest and apoptosis was assessed by oligonucleotide microarray, real-time RT-PCR and Western blot. It was shown that the treatment of KYSE-510 cells with these compounds caused G(2)/M arrest through up-regulation of p21(waf1) and down-regulation of cyclin B1 at the mRNA and protein levels, and induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3. The results of western blot analysis further showed that increases of p63 and p73 protein translation or stability might be contributed to the regulation of p21(waf1), cyclin B1 and PIG3.

Anti-Colon Cancer Effects of 6-Shogaol Through G2/M Cell Cycle Arrest by p53/p21-cdc2/cdc25A Crosstalk.

PubMed

Qi, Lian-Wen; Zhang, Zhiyu; Zhang, Chun-Feng; Anderson, Samantha; Liu, Qun; Yuan, Chun-Su; Wang, Chong-Zhi

2015-01-01

Chemopreventive agents can be identified from botanicals. Recently, there has been strong support for the potential of 6-shogaol, a natural compound from dietary ginger (Zingiber officinale), in cancer chemoprevention. However, whether 6-shogaol inhibits the growth of colorectal tumors in vivo remains unknown, and the underlying anticancer mechanisms have not been well characterized. In this work, we observed that 6-shogaol (15 mg/kg) significantly inhibited colorectal tumor growth in a xenograft mouse model. We show that 6-shogaol inhibited HCT-116 and SW-480 cell proliferation with IC50 of 7.5 and 10 μM, respectively. Growth of HCT-116 cells was arrested at the G2/M phase of the cell cycle, primarily mediated by the up-regulation of p53, the CDK inhibitor p21(waf1/cip1) and GADD45α, and by the down-regulation of cdc2 and cdc25A. Using p53(-/-) and p53(+/+) HCT-116 cells, we confirmed that p53/p21 was the main pathway that contributed to the G2/M cell cycle arrest by 6-shogaol. 6-Shogaol induced apoptosis, mainly through the mitochondrial pathway, and the bcl-2 family might act as a key regulator. Our results demonstrated that 6-shogaol induces cancer cell death by inducing G2/M cell cycle arrest and apoptosis. 6-Shogaol could be an active natural product in colon cancer chemoprevention.

G1 arrest induction represents a critical determinant for cisplatin cytotoxicity in G1 checkpoint-retaining human cancers.

PubMed

Un, Frank

2007-04-01

Cisplatin has been used effectively to treat various human cancer types; yet, the precise mechanism underlying its cytotoxicity remains unknown. In eukaryotes, progression through G1 is monitored by a checkpoint, which executes G1 arrest in the event of DNA damage to allow time for repair before initiating DNA replication. The retinoblastoma tumor suppressor gene is an integral component of the mammalian G1 checkpoint. The utility of the retinoblastoma gene as a therapeutic for human cancers has been investigated. Intriguingly, the cytotoxicity profile of the retinoblastoma gene therapy closely parallels the clinical targets of cisplatin. It prompted an investigation into the potential role of the checkpoint-induced G1 arrest in cisplatin cytotoxicity. Here, the evidence that G1 arrest induction represents a critical step in cisplatin-induced lytic path is presented. First, cisplatin-treated human cancer cells undergo a prolonged G1 arrest before dying. Second, triggering G1 arrest via infection with a recombinant adenovirus expressing the human retinoblastoma gene is sufficient to potentiate lethality in the absence of cisplatin. Third, the extent of the lethality induced correlates with the G1-arresting potential of the ectopically expressed human retinoblastoma polypeptide. Fourth, human cancer cells resistant to cisplatin do not undergo G1 arrest despite cisplatin treatment. The above mechanism may be exploited to develop therapeutics that preserve the efficacy of cisplatin yet bypass its mutagenicity associated with the formation of secondary tumors.

Symmetry of initial cell divisions among primitive hematopoietic progenitors is independent of ontogenic age and regulatory molecules.

PubMed

Huang, S; Law, P; Francis, K; Palsson, B O; Ho, A D

1999-10-15

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34(+)/CD38(-) cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% +/- 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% +/- 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34(+)/CD38(-) cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

Cell cycle arrest in plants: what distinguishes quiescence, dormancy and differentiated G1?

PubMed

Velappan, Yazhini; Signorelli, Santiago; Considine, Michael J

2017-10-17

Quiescence is a fundamental feature of plant life, which enables plasticity, renewal and fidelity of the somatic cell line. Cellular quiescence is defined by arrest in a particular phase of the cell cycle, typically G1 or G2; however, the regulation of quiescence and proliferation can also be considered across wider scales in space and time. As such, quiescence is a defining feature of plant development and phenology, from meristematic stem cell progenitors to terminally differentiated cells, as well as dormant or suppressed seeds and buds. While the physiology of each of these states differs considerably, each is referred to as 'cell cycle arrest' or 'G1 arrest'. Here the physiology and molecular regulation of (1) meristematic quiescence, (2) dormancy and (3) terminal differentiation (cell cycle exit) are considered in order to determine whether and how the molecular decisions guiding these nuclear states are distinct. A brief overview of the canonical cell cycle regulators is provided, and the genetic and genomic, as well as physiological, evidence is considered regarding two primary questions: (1) Are the canonical cell cycle regulators superior or subordinate in the regulation of quiescence? (2) Are these three modes of quiescence governed by distinct molecular controls? Meristematic quiescence, dormancy and terminal differentiation are each predominantly characterized by G1 arrest but regulated distinctly, at a level largely superior to the canonical cell cycle. Meristematic quiescence is intrinsically linked to non-cell-autonomous regulation of meristem cell identity, and particularly through the influence of ubiquitin-dependent proteolysis, in partnership with reactive oxygen species, abscisic acid and auxin. The regulation of terminal differentiation shares analogous features with meristematic quiescence, albeit with specific activators and a greater role for cytokinin signalling. Dormancy meanwhile appears to be regulated at the level of chromatin accessibility, by Polycomb group-type histone modifications of particular dormancy genes. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

NF-κB and JNK mediated apoptosis and G0/G1 arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

PubMed

Zeng, Guang-Zhi; Wang, Zhe; Zhao, Li-Mei; Fan, Jun-Ting; Tan, Ning-Hua

2018-06-28

Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. To investigate the cytotoxic mechanism of RG on cancer cells. RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G 0 /G 1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G 0 /G 1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G 0 /G 1 arrest in HeLa cells. These results indicated that RG induces mitochondria-mediated apoptosis and G 0 /G 1 cell cycle arrest by activation of JNK signaling as well as inactivation of NF-κB pathway in HeLa cells, which suggests that RG is one of the key active ingredients accounting for the anti-tumor effect of R. yunnanensis. Copyright © 2017 Elsevier B.V. All rights reserved.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

PubMed

Ottolini, Christian S; Kitchen, John; Xanthopoulou, Leoni; Gordon, Tony; Summers, Michael C; Handyside, Alan H

2017-08-29

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

PubMed Central

Alvero, Ayesha B; Visintin, Irene

2011-01-01

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer. PMID:21623171

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis*

PubMed Central

Wong, Ming-Kin; Guan, Daogang; Ng, Kaoru Hon Chun; Ho, Vincy Wing Sze; An, Xiaomeng; Li, Runsheng; Ren, Xiaoliang

2016-01-01

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development. PMID:27056332

Alignment of cell division axes in directed epithelial cell migration

NASA Astrophysics Data System (ADS)

Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

2014-11-01

Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

PubMed Central

Benanti, Jennifer A.; Galloway, Denise A.

2004-01-01

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1-transfected Chinese hamster ovary cells.

PubMed

Seo, Bo-Ra; Lee, Sook-Jeong; Cho, Kyung Sook; Yoon, Young Hee; Koh, Jae-Young

2015-12-01

Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate-conjugated Aβ(1-42) was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

Studies on the effect of cell cycle arrest on central metabolism in the diatom Phaeodactylum tricornutum, using physiological and systems biology approaches

NASA Astrophysics Data System (ADS)

Kim, Joomi

Diatoms (Bacillarophyceae) are photosynthetic unicellular microalgae that have risen to ecological prominence in the modern oceans over the past 30 million years. They are excellent candidates for biodiesel feedstocks. Global climate change has led to an interest in algal triacylglycerols (TAGs) as feedstocks for sustainable biodiesel, and diatoms are attractive candidates for TAG production as one of the most productive and environmentally flexible algae in the contemporary oceans. For Chapter 2, a genome-scale metabolic model was constructed to calculate intracellular fluxes of a diatom under different growth conditions. The model identified enzymes that may be relevant to increasing lipid synthesis, explored how transporters affect flux outputs, and explored unusual features of diatoms, including the Entner-Douderoff and phosphoketolase pathways, and glycolytic enzymes in their mitochondria. Chapter 3 discusses how cell cycle arrest via cyclin-dependent kinase (Cdk) inhibition, can increase accumulation of TAGs, and shift metabolism away from protein synthesis. For Chapter 4, transcriptome analysis of cells under cell cycle arrest was performed to show that the pattern of gene expression was fundamentally different from nitrogen stress. Most of the genes related to fatty acid and TAG synthesis were up-regulated. The gene expression pattern for light harvesting complexes was similar to cells stressed by high light, suggesting that arrested cells have smaller sinks for photosynthetically generated electrons.

Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

PubMed

Tassan, Jean-Pierre; Wühr, Martin; Hatte, Guillaume; Kubiak, Jacek

2017-01-01

Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

Triggered by asphyxia neurogenesis seems not to be an endogenous repair mechanism, gliogenesis more like it.

PubMed

Keilhoff, G; John, R; Langnaese, K; Schweizer, H; Ebmeyer, U

2010-12-15

We analyzed the long-term consequences of asphyxial cardiac arrest for hippocampal cell proliferation in rats to evaluate if the ischaemia-induced degenerated CA1 region may be repopulated by endogenous (stem) cells. Studies were performed in an asphyxial cardiac arrest model with 5 minutes of asphyxiation and three different survival times: 7, 21, and 90 days. Sham-operated non-asphyxiated rats served as control. Cell proliferation was studied by labeling dividing cells with 5-bromo-2'-deoxy-uridine (BrdU). The neurodegenerative/regenerative pattern at single cell levels was monitored by immunohistochemistry. Alterations of gene expression were analyzed by real-time quantitative RT-PCR. Analysis of BrdU-incorporation demonstrated an increase at 7, 21 as well as 90 days after global ischaemia in the hippocampal CA1 pyramidal cell layer. Similar results were found in the dentate gyrus. Differentiation of BrdU-positive cells, investigated by cell phenotype-specific double fluorescent labeling, showed increased neurogenesis only in the dentate gyrus of animals surviving the cardiac arrest for 7 days. The majority of newcomers, especially in the damaged CA1 region, consisted of glial cells. Moreover, asphyxia seemed to be able to induce the migration of microglia and astroglia from adjacent areas into the damaged area and/or the activation of resident cells. In addition, we show microglia proliferation/activation even 90 days after cardiac arrest. This morphological finding was confirmed by PCR analysis. The results indicate that asphyxia triggers cell proliferation in general and gliogenesis in particular - a possible pro-reparative event. Furthermore, from the finding of microglia proliferation up to 90 days after insult we conclude that delayed cell death processes take place which should be considered for further therapy strategies. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP.

PubMed

Beilharz, Katrin; Nováková, Linda; Fadda, Daniela; Branny, Pavel; Massidda, Orietta; Veening, Jan-Willem

2012-04-10

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Chromosome segregation drives division site selection in Streptococcus pneumoniae.

PubMed

van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

2017-07-18

Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae (pneumococcus) is an oval-shaped, symmetrically dividing opportunistic human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus, the question remains as to what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, before FtsZ. Interestingly, Z-ring formation occurs coincidently with initiation of DNA replication. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting, or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation, and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells.

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

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

Hu, Xiaolan, E-mail: huxiaolan1998@yahoo.com.cn; Zhang, Xianqi; Qiu, Shuifeng

2010-07-16

Research highlights: {yields} Salidroside inhibits the growth of human breast cancer cells. {yields} Salidroside induces cell-cycle arrest of human breast cancer cells. {yields} Salidroside induces apoptosis of human breast cancer cell lines. -- Abstract: Recently, salidroside (p-hydroxyphenethyl-{beta}-D-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) weremore » sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.« less

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.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

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

Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa

2015-04-17

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cellmore » proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.« less

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

PubMed Central

Li, Yubing; Liu, Dianyi; López-Paz, Cristina; Olson, Bradley JSC; Umen, James G

2016-01-01

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. DOI: http://dx.doi.org/10.7554/eLife.10767.001 PMID:27015111

A crucial step in cell division identified | Center for Cancer Research

Cancer.gov

When cell division doesn’t go according to plan, the resulting daughter cells can become unstable or even cancerous. A team of CCR investigators has now discovered a crucial step required for normal cell division to occur. Read more...

Interplay between cell cycle and autophagy induced by boswellic acid analog

PubMed Central

Pathania, Anup S.; Guru, Santosh K.; Kumar, Suresh; Kumar, Ashok; Ahmad, Masroor; Bhushan, Shashi; Sharma, Parduman R.; Mahajan, Priya; Shah, Bhahwal A.; Sharma, Simmi; Nargotra, Amit; Vishwakarma, Ram; Korkaya, Hasan; Malik, Fayaz

2016-01-01

In this study, we investigated the role of autophagy induced by boswellic acid analog BA145 on cell cycle progression in pancreatic cancer cells. BA145 induced robust autophagy in pancreatic cancer cell line PANC-1 and exhibited cell proliferation inhibition by inducing cells to undergo G2/M arrest. Inhibition of G2/M progression was associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Pre-treatment of cells with autophagy inhibitors or silencing the expression of key autophagy genes abrogated BA145 induced G2/M arrest and downregulation of cell cycle regulatory proteins. It was further observed that BA145 induced autophagy by targeting mTOR kinase (IC50 1 μM), leading to reduced expression of p-mTOR, p-p70S6K (T389), p-4EBP (T37/46) and p-S6 (S240/244). Notably, inhibition of mTOR signalling by BA145 was followed by attendant activation of AKT and its membrane translocation. Inhibition of Akt through pharmacological inhibitors or siRNAs enhanced BA145 mediated autophagy, G2/M arrest and reduced expression of G2/M regulators. Further studies revealed that BA145 arbitrated inhibition of mTOR led to the activation of Akt through IGFR/PI3k/Akt feedback loop. Intervention in IGFR/PI3k/Akt loop further depreciated Akt phosphorylation and its membrane translocation that culminates in augmented autophagy with concomitant G2/M arrest and cell death. PMID:27680387

Mechanism of Action of Novel Antiproliferative Oligonucleotides

DTIC Science & Technology

2002-05-01

DNA replication , cell cycle regulation, and apoptosis, the overall goal of this study was to identify the functions of nucleolin that are affected by GRO binding. After the first year of this study, several significant results have emerged. We have shown that GROs cause cell cycle arrest and induce apoptosis in prostate cancer cells but not normal skin cells, and that this arrest is due to specific inhibition of DNA replication . We have further shown that the inhibition of DNA replication may be linked to the ability of GROs to

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

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.

An in silico investigation into the causes of telomere length heterogeneity and its implications for the Hayflick limit.

PubMed

Golubev, A; Khrustalev, S; Butov, A

2003-11-21

In telomerase-negative cell populations the mean telomere length (TL) decreases with increasing population doubling number (PD). A critically small TL is believed to stop cell proliferation at a cell-, age- and species-specific PD thus defining the Hayflick limit. However, positively skewed TL distributions are broad compared to differences between initial and final mean TL and strongly overlap at middle and late PD, which is inconsistent with a limiting role of TL. We used computer-assisted modelling to define what set of premises may account for the above. Our model incorporates the following concepts. DNA end replication problem: telomeres loose 1 shortening unit (SU) upon each cell division. Free radical-caused TL decrease: telomeres experience random events resulting in the loss of a random SU number within a remaining TL. Stochasticity of gene expression and cell differentiation: cells experience random events inducing mitoses or committing cells to proliferation arrest, the latter option requiring a specified number of mitoses to be passed. Cells whose TL reaches 1SU cannot divide. The proliferation kinetics of such virtual cells conforms to the transition probability model of cell cycle. When no committing events occur and at realistic SU estimates of the initial TL, maximal PD values far exceed the Hayflick limit observed in normal cells and are consistent with the crisis stage entered by transformed cells that have surpassed the Hayflick limit. At intermediate PD, symmetrical TL distributions are yielded. Upon introduction of committing events making the ratio of the rates of proliferating and committing events (P/C) range from 1.10 to 1.25, TL distributions at intermediate PD become positively skewed, and virtual cell clones show bimodal size distributions. At P/C as high as 1.25 the majority of virtual cells at maximal PD contain telomeres with TL>1SU. A 10% increase in P/C within the 1.10-1.25 range produces a two-fold increase in the maximal PD, which can reach values of up to 25 observed in rodent and some human cells. Increasing the number of committed mitoses from 0 to 10 can increases PD to about 50 observed in human fibroblasts. Introduction of the random TL breakage makes the shapes of TL distributions quite dissimilar from those observed in real cells. Telomere length decrease is a correlate of cell proliferation that cannot alone account for the Hayflick limit, which primarily depends on parameters of cell population kinetics. Free radical damage influences the Hayflick limit not through TL but rather by affecting the ratio of the rates of events that commit cells to mitoses or to proliferation arrest.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

PubMed Central

2010-01-01

Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models. PMID:20682067

All Tumor Cells Are Not Created Equal | Center for Cancer Research

Cancer.gov

Cell division is commonly thought of as a process whereby one cell gives rise to two identical daughter cells. However, rare cell divisions are asymmetric, generating daughter cells that may differ in size, developmental potential, or even DNA content. The ability of stem cells to undergo asymmetric division allows them to self-renew while simultaneously generate daughter

Mechanical Forces Program the Orientation of Cell Division during Airway Tube Morphogenesis.

PubMed

Tang, Zan; Hu, Yucheng; Wang, Zheng; Jiang, Kewu; Zhan, Cheng; Marshall, Wallace F; Tang, Nan

2018-02-05

Oriented cell division plays a key role in controlling organogenesis. The mechanisms for regulating division orientation at the whole-organ level are only starting to become understood. By combining 3D time-lapse imaging, mouse genetics, and mathematical modeling, we find that global orientation of cell division is the result of a combination of two types of spindles with distinct spindle dynamic behaviors in the developing airway epithelium. Fixed spindles follow the classic long-axis rule and establish their division orientation before metaphase. In contrast, rotating spindles do not strictly follow the long-axis rule and determine their division orientation during metaphase. By using both a cell-based mechanical model and stretching-lung-explant experiments, we showed that mechanical force can function as a regulatory signal in maintaining the stable ratio between fixed spindles and rotating spindles. Our findings demonstrate that mechanical forces, cell geometry, and oriented cell division function together in a highly coordinated manner to ensure normal airway tube morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner

PubMed Central

Klumpp, Dominik; Misovic, Milan; Szteyn, Kalina; Shumilina, Ekaterina; Rudner, Justine; Huber, Stephan M.

2016-01-01

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation. PMID:26839633

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

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

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age, massive programmed cell death, and the release of transcriptional gene silencing. Our data suggests that plants can initiate programmed cell death to eliminate damaged cells despite the absence of p53 in plant genome.« less

Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles.

PubMed

Coelho, Paula A; Bury, Leah; Sharif, Bedra; Riparbelli, Maria G; Fu, Jingyan; Callaini, Giuliano; Glover, David M; Zernicka-Goetz, Magdalena

2013-12-09

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Cyc17, a meiosis-specific cyclin, is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila.

PubMed

Yan, Guan-Xiong; Dang, Huai; Tian, Miao; Zhang, Jing; Shodhan, Anura; Ning, Ying-Zhi; Xiong, Jie; Miao, Wei

2016-07-17

Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17.

Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation.

PubMed

García-Gómez, Candela; Parages, María L; Jiménez, Carlos; Palma, Armando; Mata, M Teresa; Segovia, María

2012-09-01

Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F(v)/F(m) and the formation of cyclobutane-pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms.

The Dietary Flavonoid Fisetin Causes Cell Cycle Arrest, Caspase-Dependent Apoptosis, and Enhanced Cytotoxicity of Chemotherapeutic Drugs in Triple-Negative Breast Cancer Cells.

PubMed

Smith, Matthew L; Murphy, Kaylee; Doucette, Carolyn D; Greenshields, Anna L; Hoskin, David W

2016-08-01

Fisetin (3,3',4',7-tetrahydroxyflavone), a flavonoid found in a number of fruits and vegetables, has diverse biological activities, including cytotoxic effects on cancer cells. In this study, we investigated the effect of fisetin on triple-negative breast cancer (TNBC) cells. TNBC has a poorer prognosis than other types of breast cancer and treatment options for this disease are limited. Fisetin inhibited the growth of MDA-MB-468 and MDA-MB-231 TNBC cells, as well as their ability to form colonies, without substantially affecting the growth of non-malignant cells. In addition, fisetin inhibited the growth of estrogen receptor-bearing MCF-7 breast cancer cells and human epidermal growth factor receptor 2-overexpressing SK-BR-3 breast cancer cells. Fisetin inhibited TNBC cell division and induced apoptosis, which was associated with mitochondrial membrane permeabilization and the activation of caspase-9 and caspase-8, as well as the cleavage of poly(ADP-ribose) polymerase-1. Induction of caspase-dependent apoptosis by fisetin was confirmed by reduced killing of TNBC cells in the presence of the pan-caspase inhibitors Z-VAD-FMK and BOC-D-FMK. Decreased phosphorylation of histone H3 at serine 10 in fisetin-treated TNBC cells at G2/M phase of the cell cycle suggested that fisetin-induced apoptosis was the result of Aurora B kinase inhibition. Interestingly, the cytotoxic effect of cisplatin, 5-fluorouracil, and 4-hydroxycyclophosphamide metabolite of cyclophosphamide on TNBC cells was increased in the presence of fisetin. These findings suggest that further investigation of fisetin is warranted for possible use in the management of TNBC. J. Cell. Biochem. 117: 1913-1925, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

NASA Technical Reports Server (NTRS)

Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

2000-01-01

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

Polyploidy Formation in Doxorubicin-Treated Cancer Cells Can Favor Escape from Senescence.

PubMed

Mosieniak, Grazyna; Sliwinska, Malgorzata A; Alster, Olga; Strzeszewska, Anna; Sunderland, Piotr; Piechota, Malgorzata; Was, Halina; Sikora, Ewa

2015-12-01

Cancer cells can undergo stress-induced premature senescence, which is considered to be a desirable outcome of anticancer treatment. However, the escape from senescence and cancer cell repopulation give rise to some doubts concerning the effectiveness of the senescence-induced anticancer therapy. Similarly, it is postulated that polyploidization of cancer cells is connected with disease relapse. We postulate that cancer cell polyploidization associated with senescence is the culprit of atypical cell divisions leading to cancer cell regrowth. Accordingly, we aimed to dissociate between these two phenomena. We induced senescence in HCT 116 cells by pulse treatment with doxorubicin and observed transiently increased ploidy, abnormal nuclear morphology, and various distributions of some proteins (e.g., p21, Ki-67, SA-β-galactosidase) in the subnuclei. Doxorubicin-treated HCT 116 cells displayed an increased production of reactive oxygen species (ROS) possibly caused by an increased amount of mitochondria, which are characterized by low membrane potential. A decrease in the level of ROS by Trolox partially protected the cells from polyploidization but not from senescence. Interestingly, a decreased level of ROS prevented the cells from escaping senescence. We also show that MCF7 cells senesce, but this is not accompanied by the increase of ploidy upon doxorubicin treatment. Moreover, they were stably growth arrested, thus proving that polyploidy but not senescence per se enables to regain the ability to proliferate. Our preliminary results indicate that the different propensity of the HCT 116 and MCF7 cells to increase ploidy upon cell senescence could be caused by a different level of the mTOR and/or Pim-1 kinases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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.

RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway

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

Wang, Haihe; Yang, Zhanchun; Liu, Chunbo

2014-11-07

Highlights: • RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. • RITA can significantly inhibit the in vitro growth of SMMC7721 and HepG2 cells. • RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC. - Abstract: Aberrant Notch signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell growth. However, the role of Notch signaling in HCC and its underlying mechanism remain elusive.more » RBP-J-interacting and tubulin-associated (RITA) mediates the nuclear export of RBP-J to tubulin fibers and downregulates Notch-mediated transcription. In this study, we found that RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Our findings indicate that RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC.« less

The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

PubMed Central

Bailey, Richard G.; Turner, Robert D.; Mullin, Nic; Clarke, Nigel; Foster, Simon J.; Hobbs, Jamie K.

2014-01-01

The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division. PMID:25468333

Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

PubMed Central

Bohnert, K. Adam; Gould, Kathleen L.

2012-01-01

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state. PMID:23093943

The role of integrin α8β1 in fetal lung morphogenesis and injury

PubMed Central

Benjamin, John T.; Gaston, David C.; Halloran, Brian A.; Schnapp, Lynn M.; Zent, Roy; Prince, Lawrence S.

2009-01-01

Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1, is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggests that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD. PMID:19769957

Chronic Exposure to Zinc Chromate Induces Centrosome Amplification and Spindle Assembly Checkpoint Bypass in Human Lung Fibroblasts

PubMed Central

Holmes, Amie L.; Wise, Sandra S.; Pelsue, Stephen C.; Aboueissa, AbouEl-Makarim; Lingle, Wilma; Salisbury, Jeffery; Gallagher, Jamie; Wise, John Pierce

2010-01-01

Hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Solubility plays an important role in its carcinogenicity with the particulate or insoluble form being the most potent. Of the particulate Cr(VI) compounds, zinc chromate appears to be the most potent carcinogen, however, very few studies have investigated its carcinogenic mechanism. In this study, we investigated the ability of chronic exposure to zinc chromate to induce numerical chromosome instability. We found no increase in aneuploidy after a 24 hour exposure to zinc chromate, but with more chronic exposures, zinc chromate induced concentration- and time-dependent increases in aneuploidy in the form of hypodiploidy, hyperdiploidy and tetraploidy. Zinc chromate also induced centrosome amplification in a concentration- and time-dependent manner in both interphase and mitotic cells after chronic exposure, producing cells with centriolar defects. Further, chronic exposure to zinc chromate induced concentration- and time-dependent increases in spindle assembly checkpoint bypass with increases in centromere spreading, premature centromere division and premature anaphase. Lastly, we found that chronic exposure to zinc chromate induced a G2 arrest. All together, these data indicate that zinc chromate can induce chromosome instability after prolonged exposures. PMID:20030412

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate.

PubMed

Saldanha, Sabita N; Kala, Rishabh; Tollefsbol, Trygve O

2014-05-15

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

PubMed Central

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest. PMID:21673927

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

Chan, R.K.; Otte, C.A.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by ..cap alpha.. factor pheromone. When sst1 mutants were mixed with normal SST/sup +/ cells, the entire population recovered together from ..cap alpha.. factor arrest, suggesting that SST/sup +/ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a ''barrier'' to the diffusion of ..cap alpha.. factor, were lesions in the same genes. These findings suggest that sst1 mutants are defective in recoverymore » from ..cap alpha.. factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST/sup +/ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to ..cap alpha.. factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of ..cap alpha.. factor for a much longer time than SST/sup +/ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of ..cap alpha.. factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective (''sterile'').« less

Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells

PubMed Central

Jin, Soojung; Park, Hyun-Jin; Oh, You Na; Kwon, Hyun Ju; Kim, Jeong-Hwan; Choi, Yung Hyun; Kim, Byung Woo

2015-01-01

Background: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). Methods: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. Results: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. Conclusions: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry. PMID:26734586

Acetate supplementation induces growth arrest of NG2/PDGFRα-positive oligodendroglioma-derived tumor-initiating cells.

PubMed

Long, Patrick M; Tighe, Scott W; Driscoll, Heather E; Moffett, John R; Namboodiri, Aryan M A; Viapiano, Mariano S; Lawler, Sean E; Jaworski, Diane M

2013-01-01

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.

Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

PubMed Central

Long, Patrick M.; Tighe, Scott W.; Driscoll, Heather E.; Moffett, John R.; Namboodiri, Aryan M. A.; Viapiano, Mariano S.; Lawler, Sean E.; Jaworski, Diane M.

2013-01-01

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation. PMID:24278309

Changes of constituents and activity to apoptosis and cell cycle during fermentation of tea.

PubMed

Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-Kun; Sheng, Jun; Shi, Wei

2011-01-01

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.

Scorpion (Androctonus crassicauda) venom limits growth of transformed cells (SH-SY5Y and MCF-7) by cytotoxicity and cell cycle arrest.

PubMed

Zargan, Jamil; Sajad, Mir; Umar, Sadiq; Naime, Mohammad; Ali, Shakir; Khan, Haider A

2011-08-01

The purpose of study was to examine the cytotoxic and anti-cancer properties along with addressing the plausible pathway followed by scorpion venom to reduce cell viability in SH-SY5Y and MCF-7 cells. Following exposure of cells with scorpion venom, cytotoxicity was estimated using MTT and lactate dehydrogenase assays. Apoptotic effects were measured by assessment of mitochondrial membrane potential, reactive nitrogen species, DNA fragmentation, and caspase-3 activity whereas antiproliferative effect was assayed using BrdU incorporation. Our results indicate that scorpion venom causes suppression of proliferation by arresting S-phase and induction of apoptosis through increased nitric oxide production, caspase-3 activity and depolarization of mitochondrial membrane. Induction of apoptosis and arrest of DNA synthesis are critical determinant factors for development of anti cancer drugs. These properties may lead to isolation of effective molecule(s) with potential anticancer activity from scorpion venom of Androctonus crassicauda. Copyright © 2011 Elsevier Inc. All rights reserved.

Cell division plane orientation based on tensile stress in Arabidopsis thaliana

PubMed Central

Louveaux, Marion; Julien, Jean-Daniel; Mirabet, Vincent; Boudaoud, Arezki; Hamant, Olivier

2016-01-01

Cell geometry has long been proposed to play a key role in the orientation of symmetric cell division planes. In particular, the recently proposed Besson–Dumais rule generalizes Errera’s rule and predicts that cells divide along one of the local minima of plane area. However, this rule has been tested only on tissues with rather local spherical shape and homogeneous growth. Here, we tested the application of the Besson–Dumais rule to the divisions occurring in the Arabidopsis shoot apex, which contains domains with anisotropic curvature and differential growth. We found that the Besson–Dumais rule works well in the central part of the apex, but fails to account for cell division planes in the saddle-shaped boundary region. Because curvature anisotropy and differential growth prescribe directional tensile stress in that region, we tested the putative contribution of anisotropic stress fields to cell division plane orientation at the shoot apex. To do so, we compared two division rules: geometrical (new plane along the shortest path) and mechanical (new plane along maximal tension). The mechanical division rule reproduced the enrichment of long planes observed in the boundary region. Experimental perturbation of mechanical stress pattern further supported a contribution of anisotropic tensile stress in division plane orientation. Importantly, simulations of tissues growing in an isotropic stress field, and dividing along maximal tension, provided division plane distributions comparable to those obtained with the geometrical rule. We thus propose that division plane orientation by tensile stress offers a general rule for symmetric cell division in plants. PMID:27436908

All Tumor Cells Are Not Created Equal | Center for Cancer Research

Cancer.gov

Cell division is commonly thought of as a process whereby one cell gives rise to two identical daughter cells. However, rare cell divisions are asymmetric, generating daughter cells that may differ in size, developmental potential, or even DNA content. The ability of stem cells to undergo asymmetric division allows them to self-renew while simultaneously generate daughter cells committed to differentiating into specialized cell types.

Creating Age Asymmetry: Consequences of Inheriting Damaged Goods in Mammalian Cells.

PubMed

Moore, Darcie L; Jessberger, Sebastian

2017-01-01

Accumulating evidence suggests that mammalian cells asymmetrically segregate cellular components ranging from genomic DNA to organelles and damaged proteins during cell division. Asymmetric inheritance upon mammalian cell division may be specifically important to ensure cellular fitness and propagate cellular potency to individual progeny, for example in the context of somatic stem cell division. We review here recent advances in the field and discuss potential effects and underlying mechanisms that mediate asymmetric segregation of cellular components during mammalian cell division. Copyright © 2016 Elsevier Ltd. All rights reserved.

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

NASA Astrophysics Data System (ADS)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

Universal rule for the symmetric division of plant cells

PubMed Central

Besson, Sébastien; Dumais, Jacques

2011-01-01

The division of eukaryotic cells involves the assembly of complex cytoskeletal structures to exert the forces required for chromosome segregation and cytokinesis. In plants, empirical evidence suggests that tensional forces within the cytoskeleton cause cells to divide along the plane that minimizes the surface area of the cell plate (Errera’s rule) while creating daughter cells of equal size. However, exceptions to Errera’s rule cast doubt on whether a broadly applicable rule can be formulated for plant cell division. Here, we show that the selection of the plane of division involves a competition between alternative configurations whose geometries represent local area minima. We find that the probability of observing a particular division configuration increases inversely with its relative area according to an exponential probability distribution known as the Gibbs measure. Moreover, a comparison across land plants and their most recent algal ancestors confirms that the probability distribution is widely conserved and independent of cell shape and size. Using a maximum entropy formulation, we show that this empirical division rule is predicted by the dynamics of the tense cytoskeletal elements that lead to the positioning of the preprophase band. Based on the fact that the division plane is selected from the sole interaction of the cytoskeleton with cell shape, we posit that the new rule represents the default mechanism for plant cell division when internal or external cues are absent. PMID:21383128

Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

PubMed Central

Huang, Shengkai; Dong, Xin; Wang, Jia; Ding, Jie; Li, Yan; Li, Dongdong; Lin, Hong; Wang, Wenjie; Zhao, Mei

2018-01-01

Background Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells. PMID:29807370

EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism.

PubMed

Adams, Brian K; Cai, Jiyang; Armstrong, Jeff; Herold, Marike; Lu, Yang J; Sun, Aiming; Snyder, James P; Liotta, Dennis C; Jones, Dean P; Shoji, Mamoru

2005-03-01

In this study, we show that the novel synthetic curcumin analog, EF24, induces cell cycle arrest and apoptosis by means of a redox-dependent mechanism in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Cell cycle analysis demonstrated that EF24 causes a G2/M arrest in both cell lines, and that this cell cycle arrest is followed by the induction of apoptosis as evidenced by caspase-3 activation, phosphatidylserine externalization and an increased number of cells with a sub-G1 DNA fraction. In addition, we demonstrate that EF24 induces a depolarization of the mitochondrial membrane potential, suggesting that the compound may also induce apoptosis by altering mitochondrial function. EF24, like curcumin, serves as a Michael acceptor reacting with glutathione (GSH) and thioredoxin 1. Reaction of EF24 with these agents in vivo significantly reduced intracellular GSH as well as oxidized GSH in both the wild-type and Bcl-xL overexpressing HT29 human colon cancer cells. We therefore propose that the anticancer effect of a novel curcumin analog, EF24, is mediated in part by redox-mediated induction of apoptosis.

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana1

PubMed Central

Beemster, Gerrit T.S.; Baskin, Tobias I.

1998-01-01

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm−1 h−1) and cell division (cells cell−1 h−1). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement. PMID:9536070

An inhibitor of polyamine synthesis arrests cells at an earlier stage of G1 than does calcium deprivation.

PubMed Central

Cheetham, B F

1983-01-01

Methylglyoxal bis(guanylhydrazone) completely inhibits the induction of thymidine kinase after serum stimulation of quiescent fibroblasts only if added within 3 h after serum, whereas calcium deprivation blocks this induction up to 12 h after serum stimulation. Experiments in which one of these blocks was imposed as the other was released confirmed that cells blocked by methylglyoxal bis(guanylhydrazone) are arrested at an earlier stage in G1 than cells blocked by calcium deprivation. PMID:6843551

Knockdown of Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 inhibits cell division and increases apoptosis in gastric cancer.

PubMed

Tang, Ze; He, Gan; Xu, Jie; Zhongfu, Li

2017-05-01

Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a pleiotropic protein associated with numerous cell functions, including transcription and differentiation. The role of CITED2 has been investigated in a number of malignancies; however, the roles of this protein in gastric cancers remain unclear. Therefore, we determined the role of CITED2 in gastric cancers. Gastric cancer cell lines (MKN74, MKN28, 7901, and AGS) were used to assess CITED2 transcript levels. Messenger RNA levels were determined using quantitative polymerase chain reaction. Lentiviral vectors containing CITED2 small interfering RNA were used to knockdown CITED2 expression. Cell proliferation was assessed with fluorescent imaging and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays. Apoptosis and cell cycle stages were assessed through flow cytometry, and formation of colonies was determined using a fluorescent microscope. All cell lines tested in this study expressed CITED2. The cell line expressing the highest levels of CITED2 (MKN74) showed significant knockdown of endogenous CITED2 expression on lentiviral infection. Cell proliferation was shown to be lower in CITED2 knockdown MKN74 cells. G1/S-phase cell cycle arrest was observed on silencing of CITED2 in MKN74 cells. A significant increase in apoptosis was observed on CITED2 knock down in MKN74 cells, while colony forming ability was significantly inhibited after knock down of CITED2. CITED2 supports gastric cancer cell colony formation and proliferation while inhibiting apoptosis making it a potential gene therapy target for gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

[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.

Pathophysiology of ocular surface squamous neoplasia

PubMed Central

Gichuhi, Stephen; Ohnuma, Shin-ichi; Sagoo, Mandeep S.; Burton, Matthew J.

2014-01-01

The incidence of ocular surface squamous neoplasia (OSSN) is strongly associated with solar ultraviolet (UV) radiation, HIV and human papilloma virus (HPV). Africa has the highest incidence rates in the world. Most lesions occur at the limbus within the interpalpebral fissure particularly the nasal sector. The nasal limbus receives the highest intensity of sunlight. Limbal epithelial crypts are concentrated nasally and contain niches of limbal epithelial stem cells in the basal layer. It is possible that these are the progenitor cells in OSSN. OSSN arises in the basal epithelial cells spreading towards the surface which resembles the movement of corneo-limbal stem cell progeny before it later invades through the basement membrane below. UV radiation damages DNA producing pyrimidine dimers in the DNA chain. Specific CC → TT base pair dimer transformations of the p53 tumour-suppressor gene occur in OSSN allowing cells with damaged DNA past the G1-S cell cycle checkpoint. UV radiation also causes local and systemic photoimmunosuppression and reactivates latent viruses such as HPV. The E7 proteins of HPV promote proliferation of infected epithelial cells via the retinoblastoma gene while E6 proteins prevent the p53 tumour suppressor gene from effecting cell-cycle arrest of DNA-damaged and infected cells. Immunosuppression from UV radiation, HIV and vitamin A deficiency impairs tumour immune surveillance allowing survival of aberrant cells. Tumour growth and metastases are enhanced by; telomerase reactivation which increases the number of cell divisions a cell can undergo; vascular endothelial growth factor for angiogenesis and matrix metalloproteinases (MMPs) that destroy the intercellular matrix between cells. Despite these potential triggers, the disease is usually unilateral. It is unclear how HPV reaches the conjunctiva. PMID:25447808

The distinctive cell division interactome of Neisseria gonorrhoeae.

PubMed

Zou, Yinan; Li, Yan; Dillon, Jo-Anne R

2017-12-12

Bacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW. Using a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsA Ng were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW. Results from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.

In vivo Post-Cardiac Arrest Myocardial Dysfunction is Supported by CaMKII-Mediated Calcium Long-Term Potentiation and Mitigated by Alda-1, an Agonist of Aldehyde Dehydrogenase Type 2

PubMed Central

Downey, Peter; Zalewski, Adrian; Rubio, Gabriel R.; Liu, Jing; Homburger, Julian R.; Grunwald, Zachary; Qi, Wei; Bollensdorff, Christian; Thanaporn, Porama; Ali, Ayyaz; Riemer, Kirk; Kohl, Peter; Mochly-Rosen, Daria; Gerstenfeld, Edward; Large, Stephen; Ali, Ziad; Ashley, Euan

2016-01-01

Background Survival after sudden cardiac arrest is limited by post-arrest myocardial dysfunction but understanding of this phenomenon is constrained by lack of data from a physiological model of disease. In this study, we established an in vivo model of cardiac arrest and resuscitation, characterized the biology of the associated myocardial dysfunction, and tested novel therapeutic strategies. Methods We developed rodent models of in vivo post-arrest myocardial dysfunction using extra-corporeal membrane oxygenation (ECMO) resuscitation followed by invasive hemodynamics measurement. In post-arrest isolated cardiomyocytes, we assessed mechanical load and Ca2+ induced Ca2+ release (CICR) simultaneously using the micro-carbon-fiber technique and observed reduced function and myofilament calcium sensitivity. We used a novel-designed fiber optic catheter imaging system, and a genetically encoded calcium sensor GCaMP6f, to image CICR in vivo. Results We found potentiation of CICR in isolated cells from this ECMO model and also in cells isolated from an ischemia-reperfusion Langendorff model perfused with oxygenated blood from an arrested animal, but not when reperfused in saline. We established that CICR potentiation begins in vivo. The augmented CICR observed post-arrest was mediated by the activation of Ca2+/calmodulin kinase II (CaMKII). Increased phosphorylation of CaMKII, phospholamban and ryanodine receptor 2 (RyR2) was detected in the post-arrest period. Exogenous adrenergic activation in vivo recapitulated Ca2+ potentiation but was associated with lesser CaMKII activation. Since oxidative stress and aldehydic adduct formation were high post arrest, we tested a small molecule activator of aldehyde dehydrogenase type 2, Alda-1, which reduced oxidative stress, restored calcium and CaMKII homeostasis, and improved cardiac function and post-arrest outcome in vivo. Conclusions Cardiac arrest and reperfusion lead to CaMKII activation and calcium long-term potentiation which support cardiomyocyte contractility in the face of impaired post-ischemic myofilament calcium sensitivity. Alda-1 mitigates these effects, normalizes calcium cycling and improves outcome. PMID:27582424

High CHMP4B expression is associated with accelerated cell proliferation and resistance to doxorubicin in hepatocellular carcinoma.

PubMed

Hu, Baoying; Jiang, Dawei; Chen, Yuyan; Wei, Lixian; Zhang, Shusen; Zhao, Fengbo; Ni, Runzhou; Lu, Cuihua; Wan, Chunhua

2015-04-01

Charged multivesicular body protein 4B (CHMP4B), a subunit of the endosomal sorting complex required for transport (ESCRT)-III complex, plays an important part in cytokinetic membrane abscission and the late stage of mitotic cell division. In this study, we explored the prognostic significance of CHMP4B in human hepatocellular carcinoma (HCC) and its impact on the physiology of HCC cells. Western blot and immunohistochemistrical analyses showed that CHMP4B was significantly upregulated in HCC tissues, compared with adjacent non-tumorous tissues. Meanwhile, clinicopathological analysis revealed that high CHMP4B expression was correlated with multiple clinicopathological variables, including AFP, cirrhosis, AJCC stage, Ki-67 expression, and poor prognosis. More importantly, univariate and multivariate survival analyses demonstrated that CHMP4B served as an independent prognostic factor for survival of HCC patients. Using HCC cell cultures, we found that the expression of CHMP4B was progressively upregulated after the release from serum starvation. To verify whether CHMP4B could regulate the proliferation of HCC cells, CHMP4B was knocked down through the transfection of CHMP4B-siRNA oligos. Flow cytometry and CCK-8 assays indicated that interference of CHMP4B led to cell cycle arrest and proliferative impairment of HCC cells. Additionally, depletion of CHMP4B expression could increase the sensitivity to doxorubicin in HepG2 and Huh7 cells. Taken together, our results implied that CHMP4B could be a promising prognostic biomarker as well as a potential therapeutic target of HCC.

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

PubMed Central

Abe, Takuya; Branzei, Dana

2014-01-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. PMID:25218467

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli.

PubMed

Fenton, Andrew K; Gerdes, Kenn

2013-07-03

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin-MreB while cell division is governed by tubulin-FtsZ. A ring-like structure containing FtsZ (the Z ring) at mid-cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid-cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB-FtsZ interaction is required for transfer of cell-wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

PubMed Central

Fenton, Andrew K; Gerdes, Kenn

2013-01-01

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring-like structure containing FtsZ (the Z ring) at mid-cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid-cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell-wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB. PMID:23756461

Cardiopulmonary Arrest and Resuscitation Disrupts Cholinergic Anti-Inflammatory Processes: A Role for Cholinergic α7 Nicotinic Receptors

PubMed Central

Morris, John S.; Karelina, Kate; Weil, Zachary M.; Zhang, Ning; Al-Abed, Yousef; Brothers, Holly M.; Wenk, Gary L.; Pavlov, Valentin A.; Tracey, Kevin J.; DeVries, A. Courtney

2011-01-01

Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1β, interleukin-6, tumor necrosis factor-α), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the α7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of α7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame. PMID:21368056

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

PubMed Central

Osawa, T; Davies, D; Hartley, J A

2011-01-01

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death. PMID:21814285

Kinetics of large-scale chromosomal movement during asymmetric cell division in Escherichia coli

PubMed Central

Männik, Jaana; O’Neill, Jordan C.

2017-01-01

Coordination between cell division and chromosome replication is essential for a cell to produce viable progeny. In the commonly accepted view, Escherichia coli realize this coordination via the accurate positioning of its cell division apparatus relative to the nucleoids. However, E. coli lacking proper positioning of its cell division planes can still successfully propagate. Here, we characterize how these cells partition their chromosomes into daughters during such asymmetric divisions. Using quantitative time-lapse imaging, we show that DNA translocase, FtsK, can pump as much as 80% (3.7 Mb) of the chromosome between daughters at an average rate of 1700±800 bp/s. Pauses in DNA translocation are rare, and in no occasions did we observe reversals at experimental time scales of a few minutes. The majority of DNA movement occurs at the latest stages of cell division when the cell division protein ZipA has already dissociated from the septum, and the septum has closed to a narrow channel with a diameter much smaller than the resolution limit of the microscope (~250 nm). Our data suggest that the narrow constriction is necessary for effective translocation of DNA by FtsK. PMID:28234902

Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells

PubMed Central

Basu, Gargi D; Pathangey, Latha B; Tinder, Teresa L; Gendler, Sandra J; Mukherjee, Pinku

2005-01-01

Introduction Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines. Methods MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model. Results The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G0/G1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and increased necrosis in the tumor mass. Conclusion The disparate molecular mechanisms of celecoxib-induced growth inhibition in human breast cancer cells depends upon the level of COX-2 expression and the invasive potential of the cell lines examined. Data suggest a role for COX-2 not only in the growth of cancer cells but also in activating the angiogenic pathway through regulating levels of vascular endothelial growth factor. PMID:15987447

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

Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

PubMed

Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

2016-01-01

Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

Development of cell-based quantitative evaluation method for cell cycle-arrest type cancer drugs for apoptosis by high precision surface plasmon resonance sensor

NASA Astrophysics Data System (ADS)

Ona, Toshihiro; Nishijima, Hiroshi; Kosaihira, Atsushi; Shibata, Junko

2008-04-01

In vitro rapid and quantitative cell-based assay is demanded to verify the efficacy prediction of cancer drugs since a cancer patient may have unconventional aspects of tumor development. Here, we show the rapid and non-label quantitative verifying method and instrumentation of apoptosis for cell cycle-arrest type cancer drugs (Roscovitine and D-allose) by reaction analysis of living liver cancer cells cultured on a sensor chip with a newly developed high precision (50 ndeg s -1 average fluctuation) surface plasmon resonance (SPR) sensor. The time-course cell reaction as the SPR angle change rate for 10 min from 30 min cell culture with a drug was significantly related to cell viability. By the simultaneous detection of differential SPR angle change and fluorescence by specific probes using the new instrument, the SPR angle was related to the nano-order potential decrease in inner mitochondrial membrane potential. The results obtained are universally valid for the cell cycle-arrest type cancer drugs, which mediate apoptosis through different cell-signaling pathways, by a liver cancer cell line of Hep G2 (P<0.001). This system towards the application to evaluate personal therapeutic potentials of drugs using cancer cells from patients in clinical use.

Salinomycin sensitizes antimitotic drugs-treated cancer cells by increasing apoptosis via the prevention of G2 arrest

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

Kim, Ju-Hwa; Yoo, Hye-In; Kang, Han Sung

Highlights: Black-Right-Pointing-Pointer Sal sensitizes antimitotic drugs-treated cancer cells. Black-Right-Pointing-Pointer Sal sensitizes them by prevention of G2 arrest and reduced cyclin D1 levels. Black-Right-Pointing-Pointer Sal also sensitizes them by increasing DNA damage and reducing p21 level. Black-Right-Pointing-Pointer A low concentration of Sal effectively sensitized the cancer cells to antimitotic drugs. -- Abstract: Here, we investigated whether Sal could sensitize cancer cells to antimitotic drugs. We demonstrated that Sal sensitized paclitaxcel (PAC)-, docetaxcel (DOC)-, vinblastin (VIN)-, or colchicine (COL)-treated cancer cell lines, suggesting that Sal has the ability to sensitize the cells to any form of microtubule-targeting drugs. Sensitization to the antimitoticmore » drugs could be achieved with very low concentrations of Sal, suggesting that there is a possibility to minimize Sal toxicity associated with human cancer patient treatments. Sensitization by Sal increased apoptosis, which was observed by C-PARP production. Sal sensitized the cancer cells to antimitotic drugs by preventing G2 arrest, suggesting that Sal contributes to the induction of mitotic catastrophe. Sal generally reduced cyclin D1 levels in PAC-, DOC-, and VIN-treated cells. In addition, Sal treatment increased pH2AX levels and reduced p21 levels in antimitotic drugs-treated cells. These observations suggest that the mechanisms underlying Sal sensitization to DNA-damaging compounds, radiation, and microtubule-targeting drugs are similar. Our data demonstrated that Sal sensitizes cancer cells to antimitotic drugs by increasing apoptosis through the prevention of G2 arrest via conserved Sal-sensitization mechanisms. These results may contribute to the development of Sal-based chemotherapy for cancer patients treated with antimitotic drugs.« less

Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells.

PubMed

Samanta, Suman K; Lee, Joomin; Hahm, Eun-Ryeong; Singh, Shivendra V

2018-07-01

We have reported previously that withaferin A (WA) prevents breast cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice, but the mechanism is not fully understood. Unbiased proteomics of the mammary tumors from control- and WA-treated MMTV-neu mice revealed downregulation of peptidyl-prolyl cis/trans isomerase (Pin1) protein by WA administration. The present study extends these findings to elucidate the role of Pin1 in cancer chemopreventive mechanisms of WA. The mammary tumor level of Pin1 protein was lower by about 55% in WA-treated rats exposed to N-methyl-N-nitrosourea, compared to control. Exposure of MCF-7 and SK-BR-3 human breast cancer cells to WA resulted in downregulation of Pin1 protein. Ectopic expression of Pin1 attenuated G 2 and/or mitotic arrest resulting from WA treatment in both MCF-7 and SK-BR-3 cells. WA-induced apoptosis was increased by Pin1 overexpression in MCF-7 cells but not in the SK-BR-3 cell line. In addition, molecular docking followed by mass spectrometry indicated covalent interaction of WA with cysteine 113 of Pin1. Overexpression of Pin1 C113A mutant failed to attenuate WA-induced mitotic arrest or apoptosis in the MCF-7 cells. Furthermore, antibody array revealed upregulation of proapoptotic insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, in Pin1 overexpressing MCF-7 cells following WA treatment when compared to empty vector transfected control cells. These data support a crucial role of the Pin1 for mitotic arrest and apoptosis signaling by WA at least in the MCF-7 cells. © 2018 Wiley Periodicals, Inc.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

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

Carrasco-Garcia, Estefania; Saceda, Miguel; Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cellmore » lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.« less

Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25.

PubMed

Huard, Sylvain; Elder, Robert T; Liang, Dong; Li, Ge; Zhao, Richard Y

2008-03-01

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Adiposity Alters Genes Important in Inflammation and Cell Cycle Division in Human Cumulus Granulosa Cell.

PubMed

Merhi, Zaher; Polotsky, Alex J; Bradford, Andrew P; Buyuk, Erkan; Chosich, Justin; Phang, Tzu; Jindal, Sangita; Santoro, Nanette

2015-10-01

To determine whether obesity alters genes important in cellular growth and inflammation in human cumulus granulosa cells (GCs). Eight reproductive-aged women who underwent controlled ovarian hyperstimulation followed by oocyte retrieval for in vitro fertilization were enrolled. Cumulus GC RNA was extracted and processed for microarray analysis on Affymetrix Human Genome U133 Plus 2.0 chips. Gene expression data were validated on GCs from additional biologically similar samples using quantitative real-time polymerase chain reaction (RT-PCR). Comparison in gene expression was made between women with body mass index (BMI) <25 kg/m(2) (group 1; n = 4) and those with BMI ≥25 kg/m(2) (group 2; n = 4). Groups 1 and 2 had significantly different BMI (21.4 ± 1.4 vs 30.4 ± 2.7 kg/m(2), respectively; P = .02) but did not differ in age (30.5 ± 1.7 vs 32.7 ± 0.3 years, respectively; P = .3). Comparative analysis of gene expression profiles by supervised clustering between group 1 versus group 2 resulted in the selection of 7 differentially expressed genes: fibroblast growth factor 12 (FGF-12), protein phosphatase 1-like (PPM1L), zinc finger protein multitype 2 (ZFPM2), forkhead box M1 (FOXM1), cell division cycle 20 (CDC20), interleukin 1 receptor-like 1 (IL1RL1), and growth arrest-specific protein 7 (GAS7). FOXM1, CDC20, and GAS7 were downregulated while FGF-12 and PPM1L were upregulated in group 2 when compared to group 1. Validation with RT-PCR confirmed the microarray data except for ZFPM2 and IL1RL. As BMI increased, expression of FOXM1 significantly decreased (r = -.60, P = .048). Adiposity is associated with changes in the expression of genes important in cellular growth, cell cycle progression, and inflammation. The upregulation of the metabolic regulator gene PPM1L suggests that adiposity induces an abnormal metabolic follicular environment, potentially altering folliculogenesis and oocyte quality. © The Author(s) 2015.

Adiposity Alters Genes Important in Inflammation and Cell Cycle Division in Human Cumulus Granulosa Cell

PubMed Central

Merhi, Zaher; Polotsky, Alex J.; Bradford, Andrew P.; Buyuk, Erkan; Chosich, Justin; Phang, Tzu; Jindal, Sangita; Santoro, Nanette

2015-01-01

Objective: To determine whether obesity alters genes important in cellular growth and inflammation in human cumulus granulosa cells (GCs). Methods: Eight reproductive-aged women who underwent controlled ovarian hyperstimulation followed by oocyte retrieval for in vitro fertilization were enrolled. Cumulus GC RNA was extracted and processed for microarray analysis on Affymetrix Human Genome U133 Plus 2.0 chips. Gene expression data were validated on GCs from additional biologically similar samples using quantitative real-time polymerase chain reaction (RT-PCR). Comparison in gene expression was made between women with body mass index (BMI) <25 kg/m2 (group 1; n = 4) and those with BMI ≥25 kg/m2 (group 2; n = 4). Results: Groups 1 and 2 had significantly different BMI (21.4 ± 1.4 vs 30.4 ± 2.7 kg/m2, respectively; P = .02) but did not differ in age (30.5 ± 1.7 vs 32.7 ± 0.3 years, respectively; P = .3). Comparative analysis of gene expression profiles by supervised clustering between group 1 versus group 2 resulted in the selection of 7 differentially expressed genes: fibroblast growth factor 12 (FGF-12), protein phosphatase 1-like (PPM1L), zinc finger protein multitype 2 (ZFPM2), forkhead box M1 (FOXM1), cell division cycle 20 (CDC20), interleukin 1 receptor-like 1 (IL1RL1), and growth arrest-specific protein 7 (GAS7). FOXM1, CDC20, and GAS7 were downregulated while FGF-12 and PPM1L were upregulated in group 2 when compared to group 1. Validation with RT-PCR confirmed the microarray data except for ZFPM2 and IL1RL. As BMI increased, expression of FOXM1 significantly decreased (r = −.60, P = .048). Conclusions: Adiposity is associated with changes in the expression of genes important in cellular growth, cell cycle progression, and inflammation. The upregulation of the metabolic regulator gene PPM1L suggests that adiposity induces an abnormal metabolic follicular environment, potentially altering folliculogenesis and oocyte quality. PMID:25676576

Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

PubMed Central

2013-01-01

Background Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. Methods The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. Results The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Conclusions Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma. PMID:23915425