Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells

PubMed Central

Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D.; Yu, Jun; Zhu, Lihua Julie

2017-01-01

ABSTRACT The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells. PMID:28630280

Synergistic effects of the sesquiterpene lactone, EPD, with cisplatin and paclitaxel in ovarian cancer cells.

PubMed

van Haaften, Caroline; Boot, Arnoud; Corver, Willem E; van Eendenburg, Jaap D H; Trimbos, Baptist J M Z; van Wezel, Tom

2015-04-25

Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.

A stochastic spatiotemporal model of a response-regulator network in the Caulobacter crescentus cell cycle

NASA Astrophysics Data System (ADS)

Li, Fei; Subramanian, Kartik; Chen, Minghan; Tyson, John J.; Cao, Yang

2016-06-01

The asymmetric cell division cycle in Caulobacter crescentus is controlled by an elaborate molecular mechanism governing the production, activation and spatial localization of a host of interacting proteins. In previous work, we proposed a deterministic mathematical model for the spatiotemporal dynamics of six major regulatory proteins. In this paper, we study a stochastic version of the model, which takes into account molecular fluctuations of these regulatory proteins in space and time during early stages of the cell cycle of wild-type Caulobacter cells. We test the stochastic model with regard to experimental observations of increased variability of cycle time in cells depleted of the divJ gene product. The deterministic model predicts that overexpression of the divK gene blocks cell cycle progression in the stalked stage; however, stochastic simulations suggest that a small fraction of the mutants cells do complete the cell cycle normally.

Octyl gallate and gallic acid isolated from Terminalia bellarica regulates normal cell cycle in human breast cancer cell lines.

PubMed

Sales, Mary Selesty; Roy, Anita; Antony, Ludas; Banu, Sakhila K; Jeyaraman, Selvaraj; Manikkam, Rajalakshmi

2018-07-01

Herbal medicines stand unique and effective in treating human diseases. Terminalia bellarica (T. bellarica) is a potent medicinal herb, with a wide range of pharmacological activities. The present study was aimed to evaluate the effect of octyl gallate (OG) and gallic acid (GA) isolated from methanolic fruit extract of T. bellirica to inhibit the survival of breast cancer cells (MCF-7 & MDA-MB-231). Both OG & GA exhibited decreased MCF-7 & MDA-MB-231 survival and induced apoptosis, with IC 50 value of OG and GA as 40 μM and 80 μM respectively. No toxic effect was observed on normal breast cells (MCF-10A). The compounds inhibited cell cycle progression by altering the expression of the cell cycle regulators (Cyclin D1, D3, CDK-4, CDK-6, p18 INK4, p21Waf-1 and p27 KIP). Octyl gallate was more effective at low concentrations than GA. In-silico results provided stable interactions between the compounds and target proteins. The present investigation proved the downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators inducing apoptosis in compound-treated breast cancer cells. Hence, both the compounds may serve as potential anticancer agents and could be developed as breast cancer drugs, with further explorations. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

X ray sensitivity of diploid skin fibroblasts from patients with Fanconi's anemia

NASA Technical Reports Server (NTRS)

Kale, Ranjini

1989-01-01

Experiments were performed on Fanconi's anemia and normal human fibroblast cell lines growing in culture in an attempt to correlate cell cycle kinetics with genomic damage and determine their bearing on the mechanism of chromosome aberration induction. FA fibroblasts showed a significantly increased susceptibility to chromosomal breakage by x rays in the G2 phase of the cell cycle. No such response was observed in fibroblasts irradiated in the G0 phase. The observed increases in achromatic lesions and in chromatid deletions in FA cells as compared with normal cells appear to indicate that FA cells are deficient in strand break repair and also possibly in base damage excision repair. Experiments are now in progress to further elucidate the mechanisms involved.

A novel quantitative model of cell cycle progression based on cyclin-dependent kinases activity and population balances.

PubMed

Pisu, Massimo; Concas, Alessandro; Cao, Giacomo

2015-04-01

Cell cycle regulates proliferative cell capacity under normal or pathologic conditions, and in general it governs all in vivo/in vitro cell growth and proliferation processes. Mathematical simulation by means of reliable and predictive models represents an important tool to interpret experiment results, to facilitate the definition of the optimal operating conditions for in vitro cultivation, or to predict the effect of a specific drug in normal/pathologic mammalian cells. Along these lines, a novel model of cell cycle progression is proposed in this work. Specifically, it is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases experienced by each cell of the entire population during its own life. The transition between two consecutive cell cycle phases is simulated by taking advantage of the biochemical kinetic model developed by Gérard and Goldbeter (2009) which involves cyclin-dependent kinases (CDKs) whose regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylation-dephosphorylation, and cyclin synthesis or degradation. This biochemical model properly describes the entire cell cycle of mammalian cells by maintaining a sufficient level of detail useful to identify check point for transition and to estimate phase duration required by PB. Specific examples are discussed to illustrate the ability of the proposed model to simulate the effect of drugs for in vitro trials of interest in oncology, regenerative medicine and tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

PubMed Central

Benavides Damm, Tatiana; Franco-Obregón, Alfredo; Egli, Marcel

2013-01-01

Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G2/M phase progression, where cells accumulated specifically between the G2 checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading. PMID:23974110

Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle.

PubMed

Pesavento, James J; Yang, Hongbo; Kelleher, Neil L; Mizzen, Craig A

2008-01-01

Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G(2), M, and G(1) phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.

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.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cell - Update II

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1992-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV) nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles, compared to 3500 cycles for cells containing 31 percent KOH. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2X normal rate). The depth-of-discharge was 80 percent. Six 48-Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16,000 cycles during the continuing test.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

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.

Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

PubMed

Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

2014-03-30

Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts*

PubMed Central

Galindo, Mario; Pratap, Jitesh; Young, Daniel W.; Hovhannisyan, Hayk; Im, Hee-Jeong; Choi, Je-Yong; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; van Wijnen, Andre J.

2010-01-01

The Runx2 (CBFA1/AML3/PEBP2αA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.e. G0/G1 transition) in preconfluent MC3T3 osteoblastic cells that do not yet express mature bone phenotypic gene expression. Cell growth regulation of Runx2 is also observed in primary calvarial osteoblasts and other osteoblastic cells with relatively normal cell growth characteristics, but not in osteosarcoma cells (e.g. SAOS-2 and ROS17/2.8). Runx2 levels are cell cycle-regulated in MC3T3 cells with respect to the G1/S and M/G1 transitions: expression oscillates from maximal levels during early G1 to minimal levels during early S phase and mitosis. However, in normal or immortalized (e.g. ATDC5) chondrocytic cells, Runx2 expression is suppressed during quiescence, and Runx2 levels are not regulated during G1 and S phase in ATDC5 cells. Antisense or small interfering RNA-mediated reduction of the low physiological levels of Runx2 in proliferating MC3T3 cells does not accelerate cell cycle progression. However, forced expression of Runx2 suppresses proliferation of MC3T3 preosteoblasts or C2C12 mesenchymal cells which have osteogenic potential. Forced elevation of Runx2 in synchronized MC3T3 cells causes a delay in G1. We propose that Runx2 levels and function are biologically linked to a cell growth-related G1 transition in osteoblastic cells. PMID:15781466

High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development

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

Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui

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

7 CFR 1794.23 - Proposals normally requiring an EA.

Code of Federal Regulations, 2011 CFR

2011-01-01

... classification are: (1) Construction of fuel cell, combustion turbine, combined cycle, or diesel generating... be covered in the EA; (2) Construction of fuel cell, combustion turbine, combined cycle, or diesel... boundaries. (12) Installing a heat recovery steam generator and steam turbine with a rating of more than 200...

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

PubMed Central

2011-01-01

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

Nox2-derived ROS in PPARγ signaling and cell-cycle progression of lung alveolar epithelial cells

PubMed Central

Tickner, Jennifer; Fan, Lampson M.; Du, Junjie; Meijles, Daniel; Li, Jian-Mei

2011-01-01

Reactive oxygen species (ROS) play important roles in peroxisome proliferator-activated receptor γ (PPARγ) signaling and cell-cycle regulation. However, the PPARγ redox-signaling pathways in lung alveolar epithelial cells remain unclear. In this study, we investigated the in vivo and in vitro effects of PPARγ activation on the levels of lung ROS production and cell-cycle progression using C57BL/6J wild-type and Nox2 knockout mice (n = 10) after intraperitoneal injection of a selective PPARγ agonist (GW1929, 5 mg/kg body wt, daily) for 14 days. Compared to vehicle-treated mice, GW1929 increased significantly the levels of ROS production in wild-type lungs, and this was accompanied by significant up-regulation of PPARγ, Nox2, PCNA, and cyclin D1 and phosphorylation of ERK1/2 and p38MAPK. These effects were absent in Nox2 knockout mice. In cultured alveolar epithelial cells, GW1929 (5 μM for 24 h) increased ROS production and promoted cell-cycle progression from G0/G1 into S and G2/M phases, and these effects were abolished by (1) adding a PPARγ antagonist (BADGE, 1 μM), (2) knockdown of PPARγ using siRNA, or (3) knockout of Nox2. In conclusion, PPARγ activation through Nox2-derived ROS promotes cell-cycle progression in normal mouse lungs and in cultured normal alveolar epithelial cells. PMID:21664456

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

PubMed Central

Barbaric, Ivana; Biga, Veronica; Gokhale, Paul J.; Jones, Mark; Stavish, Dylan; Glen, Adam; Coca, Daniel; Andrews, Peter W.

2014-01-01

Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns. PMID:25068128

Coordination of cell death and the cell cycle: linking proliferation to death through private and communal couplers.

PubMed

Abrams, John M; White, Michael A

2004-12-01

In development and in the adult, complex signaling pathways operate within and between cells to coordinate proliferation and cell death. These networks can be viewed as coupling devices that link engines driving the cell cycle and the initiation of apoptosis. We propose three simple frameworks for modeling the effects of proliferative drive on apoptotic propensity. This perspective offers a potentially useful foundation for predicting group behaviors of cells in normal and pathological settings.

Perturbation of host-cell membrane is a primary mechanism of HIV cytopathology.

PubMed

Cloyd, M W; Lynn, W S

1991-04-01

Cytopathic viruses injure cells by a number of different mechanisms. The mechanism by which HIV-1 injures T cells was studied by temporally examining host-cell macromolecular syntheses, stages of the cell cycle, and membrane permeability following acute infection. T cells cytopathically infected at an m.o.i. of 1-5 grew normally for 24-72 hr, depending on the cell line, followed by the first manifestation of cell injury, slowing of cell division. At that time significant amounts of unintegrated HIV DNA and p24 core protein became detectable, and acridine orange flow cytometric cell cycle studies demonstrated the presence of fewer cells in the G2/M stage of the cell cycle. There was no change in the frequency of cells in the S-stage, and metabolic pulsing with radioactive precursors demonstrated that host-cell DNA, RNA, and protein syntheses were normal at that time and normal up to the time cells started to die (approximately 24 hr later), when all three decreased. Cellular lipid synthesis, however, was perturbed when cell multiplication slowed, with phospholipid synthesis reduced and neutral lipid synthesis enhanced. Permeability of the host-cell membrane to small molecules, such as Ca2+ and sucrose, was slightly enhanced early postinfection, and by the time of slowing of cell division, host membrane permeability was greatly increased to both Ca2+ and sucrose (Stokes radius 5.2 A) but not to inulin (Stokes radium 20 A). These changes in host-cell membrane permeability and phospholipid synthesis were not observed in acutely infected H9 cells, which are not susceptible to HIV cytopathology. Thus, HIV-1 appeared to predominantly injure T cells by perturbing host-cell membrane permeability and lipid synthesis, which is similar to the cytopathic mechanisms of paramyxoviruses.

Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines

PubMed Central

Taparia, Shruti; Khanna, Aparna

2016-01-01

Background: Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancer Objective: To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines. Materials and Methods: Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry. Results: The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression. Conclusion: Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells. SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized cocoa powder is one of the richest sources of procyanidinsCocoa procyanidin-rich extract (CPRE) caused cytotoxicity and chemosensitization in ovarian carcinoma cell lines OAW42 and OVCAR3CPRE affected normal cell cycle progressionCPRE also downregulated P-glycoprotein, which mediates chemoresistance in multidrug-resistant OVCAR3 cell line. Abbreviations used: P-gp: P-glycoprotein, CPRE: Cocoa procyanidin rich extract, DMAC: 4-dimethylaminocinnamaldehyde, DPPH: Diphenylpicrylhydrazyl, ABTS: 2,2’;-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), PI: Propidium iodide, FITC: Fluorescein isothiocyanate, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, TLC: Thin layer chromatography, HPTLC: High-performance thin layer chromatography. PMID:27279694

Transforming Growth Factor-B Receptors in Human Breast Cancer.

DTIC Science & Technology

1998-05-01

I., Polyak, K., Iavarone, A., and Massagud, J. Kip/ Cip and Ink4 cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-ß. Genes Dev...specimens. Thirdly, we have developped transient transfection assays to determine how specific TßR mutations affect affect receptor function. Using...Growth Factor-ß (TGFß) is the most potent known inhibitor of cell cycle progression of normal mammary epithelial cells; in addition, it causes cells

Anti-colorectal cancer effects of tripolinolate A from Tripolium vulgare.

PubMed

Chen, Lu; Wang, Wen-Ling; Song, Teng-Fei; Xie, Xin; Ye, Xue-Wei; Liang, Ying; Huang, Hao-Cai; Yan, Shi-Lun; Lian, Xiao-Yuan; Zhang, Zhi-Zhen

2017-08-01

Tripolinolate A (TLA) is recently identified as a new compound from a halophyte plant Tripolium vulgare and has been shown to have significant in vitro activity against the proliferation of colorectal cancer and glioma cells. This study was designed to further investigate the effects of TLA on the proliferation of human normal cells, and the apoptosis and cell cycle in colorectal cancer cells, and the growth of tumors in the colorectal cancer-bearing animals. The data obtained from this study demonstrated that: 1) TLA had much less cytotoxicity in the human normal cells than the colorectal cancer cells; 2) TLA remarkably induced apoptosis in the human colorectal cancer cells and blocked cell cycle at G 2 /M phase, and 3) TLA had significant anti-colorectal cancer activity in the tumor-bearing animals. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

PubMed

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

Day-night cycles and the sleep-promoting factor, Sleepless, affect stem cell activity in the Drosophila testis.

PubMed

Tulina, Natalia M; Chen, Wen-Feng; Chen, Jung Hsuan; Sowcik, Mallory; Sehgal, Amita

2014-02-25

Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. Here, we explore a role of environmental day-night cycles in modulating cell cycle progression in populations of adult stem cells. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, we reveal daily rhythms in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. We also examine whether behavioral sleep-wake cycles, which are driven by the environmental day-night cycles, regulate stem cell function. We find that flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. We suggest that alterations in sleep can influence the daily dynamics of GSC divisions.

Different responses of tumor and normal cells to low-dose radiation

PubMed Central

Liu, Ning; Wang, Hao; Shang, Qingjun; Jiang, Peng; Zhang, Yuanmei

2013-01-01

Aim of the study We demonstrated stimulation of both erythrocyte immune function and superoxide dismutase activity in tumor-bearing mice in response to whole-body 75 mGy X-rays. In addition, we enhanced the chemotherapeutic effect by exposing tumor-bearing mice to low-dose radiation (LDR). This study aims to investigate the different responses of tumor cells and normal cells to LDR. Material and methods Survival fraction, micronucleus frequency, and cell cycle of Lewis cells and primary human fibroblast AG01522 cells were measured. S180 sarcoma cells were implanted in mice, and tumor sizes were measured in vivo. Results In response to LDR exposure in vitro, a stimulating effect was observed in AG01522 cells but not in Lewis cells. Low-dose radiation did not cause an adaptive response in the Lewis cell cycle. Lack of an LDR-induced radioadaptive response in tumor cells was observed in tumor-bearing mouse models. Furthermore, a higher apoptotic effect and lower expression of the anti-apoptosis gene Bcl-2 were found in tumor cells of tumor-bearing mice exposed to D1 + D2 than those in tumor cells of tumor-bearing mice exposed to D2 alone. Conclusions Different responses of tumor cells and normal cells to LDR were found. Low-dose radiation was found to stimulate the growth of normal cells but not of tumor cells in vitro and in vivo, which is a very important and clinically relevant phenomenon. PMID:24592123

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

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

Srivastava, Janmejai K.; Department of Urology, University Hospitals of Cleveland, Cleveland, OH 44106; Gupta, Sanjay

2006-07-28

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation inmore » all three prostate cancer cell lines. The IC{sub 5} values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 {mu}g/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 {mu}g/ml. In cell cycle analysis, TRF (10-40 {mu}g/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.« less

Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity.

PubMed

Desforges, Bénédicte; Savarin, Philippe; Bounedjah, Ouissame; Delga, Stéphanie; Hamon, Loïc; Curmi, Patrick A; Pastré, David

2011-09-01

Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.

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

Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Bongso, Ariff

2010-03-01

The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.

Extremely low-frequency electromagnetic fields cause DNA strand breaks in normal cells

PubMed Central

2014-01-01

Background Extremely low frequency electromagnetic fields aren’t considered as a real carcinogenic agent despite the fact that some studies have showed impairment of the DNA integrity in different cells lines. The aim of this study was evaluation of the late effects of a 100 Hz and 5.6 mT electromagnetic field, applied continuously or discontinuously, on the DNA integrity of Vero cells assessed by alkaline Comet assay and by cell cycle analysis. Normal Vero cells were exposed to extremely low frequency electromagnetic fields (100 Hz, 5.6 mT) for 45 minutes. The Comet assay and cell cycle analysis were performed 48 hours after the treatment. Results Exposed samples presented an increase of the number of cells with high damaged DNA as compared with non-exposed cells. Quantitative evaluation of the comet assay showed a significantly (<0.001) increase of the tail lengths, of the quantity of DNA in tail and of Olive tail moments, respectively. Cell cycle analysis showed an increase of the frequency of the cells in S phase, proving the occurrence of single strand breaks. The most probable mechanism of induction of the registered effects is the production of different types of reactive oxygen species. Conclusions The analysis of the registered comet indices and of cell cycle showed that extremely low frequency electromagnetic field of 100 Hz and 5.6 mT had a genotoxic impact on Vero cells. PMID:24401758

Modelling cell cycle synchronisation in networks of coupled radial glial cells.

PubMed

Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R

2015-07-21

Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

PubMed

Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.

7 CFR 1794.25 - Proposals normally requiring an EIS.

Code of Federal Regulations, 2011 CFR

2011-01-01

... (nameplate rating) other than fuel cell, combustion turbine, combined cycle, or diesel generators. All new... water and waste programs. No groups or sets of proposed actions normally require the preparation of an...

7 CFR 1794.25 - Proposals normally requiring an EIS.

Code of Federal Regulations, 2012 CFR

2012-01-01

... (nameplate rating) other than fuel cell, combustion turbine, combined cycle, or diesel generators. All new... water and waste programs. No groups or sets of proposed actions normally require the preparation of an...

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

PubMed

Carcagno, Abel L; Marazita, Mariela C; Ogara, María F; Ceruti, Julieta M; Sonzogni, Silvina V; Scassa, María E; Giono, Luciana E; Cánepa, Eduardo T

2011-01-01

A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

PubMed Central

Carcagno, Abel L.; Marazita, Mariela C.; Ogara, María F.; Ceruti, Julieta M.; Sonzogni, Silvina V.; Scassa, María E.; Giono, Luciana E.; Cánepa, Eduardo T.

2011-01-01

Background A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. Methodology/Principal Findings In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. Conclusions/Significance The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity. PMID:21765927

NONO couples the circadian clock to the cell cycle.

PubMed

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

2013-01-29

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

Antiproliferative activity of Greek propolis.

PubMed

Pratsinis, Harris; Kletsas, Dimitris; Melliou, Eleni; Chinou, Ioanna

2010-04-01

The butanolic extract and the isolated chemical constituents, mainly diterpenes and flavonoids, from Greek propolis have been tested for their cytostatic activities against human malignant and normal cell strains. The extract and the diterpenes were found to be the most active against HT-29 human colon adenocarcinoma cells, without affecting normal human cells. Manool, a diterpene isolated for the first time from Greek propolis, was the most active compound, arresting the cancer cells at the G(2)/M phase of the cell cycle.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in low earth orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells was reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and a real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana, to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There was two failures, however, for the cells containing 31 percent KOH.

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in the low-earth-orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells is reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There were two failures, however, for the cells containing 31 percent KOH.

Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

PubMed

Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

2016-07-19

Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

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

2000-01-01

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

Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth.

PubMed

Edgar, Bruce A; Zielke, Norman; Gutierrez, Crisanto

2014-03-01

In endoreplication cell cycles, known as endocycles, cells successively replicate their genomes without segregating chromosomes during mitosis and thereby become polyploid. Such cycles, for which there are many variants, are widespread in protozoa, plants and animals. Endocycling cells can achieve ploidies of >200,000 C (chromatin-value); this increase in genomic DNA content allows a higher genomic output, which can facilitate the construction of very large cells or enhance macromolecular secretion. These cells execute normal S phases, using a G1-S regulatory apparatus similar to the one used by mitotic cells, but their capability to segregate chromosomes has been suppressed, typically by downregulation of mitotic cyclin-dependent kinase activity. Endocycles probably evolved many times, and the various endocycle mechanisms found in nature highlight the versatility of the cell cycle control machinery.

[Membrane model of the regulation of proliferation: the theory and interpretation of an experiment].

PubMed

Volkov, E I

1983-04-01

The role of cell surface physical organization in the cell cycle regulation is analyzed within the framework of the earlier proposed theory (Chernavskii et al., 1982). Two models of cell surface are considered: hard-frame fluid-mosaic model (latticemosaic) and the fluid-mosaic one. The former deals with normal cells. The existence of integral carcasse or "frame" which is formed by the essential part of cross-linked membrane components and may have at least two different conformational states is hypothesized. The second model describes membranes of tumour cells. With the latter theory any mitogen (excluding the restoration of nutrient depletion) reduces the mechanical tensile strength of the frame and stimulates the general structural rearrangement of the plasma membrane. There are only two conformational transitions during the cell cycle which serve as signals for the beginning of S and M phases. If the values of tensile strength are great enough and therefore the conformational transitions are impossible, the cells pass into the resting (prereplicative--G01, or premitotical--G02) state. Three types of experiments are interpreted in the proposed theory: a) on differences in the action of growth factors on normal and tumour cell cycle, b) on the necessary condition for mitogenicity of lectins, c) on the stimulation of proliferation by mechanical deformation of cells.

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.

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.

Circadian clock regulation of the cell cycle in the zebrafish intestine.

PubMed

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

2013-01-01

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

Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

PubMed Central

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

2013-01-01

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

Dihydroartemisinin inhibits indoxyl sulfate (IS)-promoted cell cycle progression in mesangial cells by targeting COX-2/mPGES-1/PGE2 cascade.

PubMed

Mungun, Harr-Keshauve; Li, Shuzhen; Zhang, Yue; Huang, Songming; Jia, Zhanjun; Ding, Guixia; Zhang, Aihua

2018-01-01

Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has been used as an antimalarial drug. Recently, roles of artemisinin and its derivatives in treating diseases besides antimalarial effect were documented. Thus, this study was undertaken to investigate the role of DHA in indoxyl sulfate (IS)-promoted cell cycle progression in glomerular mesangial cells, as well as the potential mechanisms. Under the basal condition, DHA significantly retarded the cell cycle progression as shown by decreased cell percentage in S phase and increased cell percentage in G1/G0 phases in line with reduced cell cycle proteins cyclin A2 and cyclin D1. Interestingly, DHA also inactivated the COX-2/mPGES-1/PGE 2 cascade which has been shown to play a critical role in promoting the mesangial cell cycle progression by our previous studies. Next, we investigated the role of DHA in IS-triggered cell cycle progression in this mesangial cell line. As expected, DHA treatment significantly retarded IS-induced cell cycle progression and inhibited the activation of COX-2/mPGES-1/PGE 2 cascade induced by IS. In summary, these data indicated that DHA inhibited the cell cycle progression in glomerular mesangial cells under normal condition or IS challenge possibly through the inhibition of COX-2/mPGES-1/PGE 2 cascade, suggesting a potential of DHA in treating glomerular diseases with mesangial cell proliferation.

Repair of oxidative DNA damage, cell-cycle regulation and neuronal death may influence the clinical manifestation of Alzheimer's disease.

PubMed

Silva, Aderbal R T; Santos, Ana Cecília Feio; Farfel, Jose M; Grinberg, Lea T; Ferretti, Renata E L; Campos, Antonio Hugo Jose Froes Marques; Cunha, Isabela Werneck; Begnami, Maria Dirlei; Rocha, Rafael M; Carraro, Dirce M; de Bragança Pereira, Carlos Alberto; Jacob-Filho, Wilson; Brentani, Helena

2014-01-01

Alzheimer's disease (AD) is characterized by progressive cognitive decline associated with a featured neuropathology (neuritic plaques and neurofibrillary tangles). Several studies have implicated oxidative damage to DNA, DNA repair, and altered cell-cycle regulation in addition to cell death in AD post-mitotic neurons. However, there is a lack of studies that systematically assess those biological processes in patients with AD neuropathology but with no evidence of cognitive impairment. We evaluated markers of oxidative DNA damage (8-OHdG, H2AX), DNA repair (p53, BRCA1, PTEN), and cell-cycle (Cdk1, Cdk4, Cdk5, Cyclin B1, Cyclin D1, p27Kip1, phospho-Rb and E2F1) through immunohistochemistry and cell death through TUNEL in autopsy hippocampal tissue samples arrayed in a tissue microarray (TMA) composed of three groups: I) "clinical-pathological AD" (CP-AD)--subjects with neuropathological AD (Braak ≥ IV and CERAD = B or C) and clinical dementia (CDR ≥ 2, IQCODE>3.8); II) "pathological AD" (P-AD)--subjects with neuropathological AD (Braak ≥ IV and CERAD = B or C) and without cognitive impairment (CDR 0, IQCODE<3.2); and III) "normal aging" (N)--subjects without neuropathological AD (Braak ≤ II and CERAD 0 or A) and with normal cognitive function (CDR 0, IQCODE<3.2). Our results show that high levels of oxidative DNA damage are present in all groups. However, significant reductions in DNA repair and cell-cycle inhibition markers and increases in cell-cycle progression and cell death markers in subjects with CP-AD were detected when compared to both P-AD and N groups, whereas there were no significant differences in the studied markers between P-AD individuals and N subjects. This study indicates that, even in the setting of pathological AD, healthy cognition may be associated with a preserved repair to DNA damage, cell-cycle regulation, and cell death in post-mitotic neurons.

Thymoquinone, a bioactive component of Nigella sativa, normalizes insulin secretion from pancreatic β-cells under glucose overload via regulation of malonyl-CoA

PubMed Central

Gray, Joshua P.; Zayasbazan Burgos, Delaine; Yuan, Tao; Seeram, Navindra; Rebar, Rebecca; Follmer, Rebecca

2015-01-01

Thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) is a major bioactive component of Nigella sativa, a plant used in traditional medicine to treat a variety of symptoms, including elevated blood glucose levels in type 2 diabetic patients. Normalization of elevated blood glucose depends on both glucose disposal by peripheral tissues and glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. We employed clonal β-cells and rodent islets to investigate the effects of thymoquinone (TQ) and Nigella sativa extracts (NSEs) on GSIS and cataplerotic metabolic pathways implicated in the regulation of GSIS. TQ and NSE regulated NAD(P)H/NAD(P)+ ratios via a quinone-dependent redox cycling mechanism. TQ content was positively correlated with the degree of redox cycling activity of NSE extracts, suggesting that TQ is a major component engaged in mediating NSE-dependent redox cycling. Both acute and chronic exposure to TQ and NSE enhanced GSIS and were associated with the ability of TQ and NSE to increase the ATP/ADP ratio. Furthermore, TQ ameliorated the impairment of GSIS following chronic exposure of β-cells to glucose overload. This protective action was associated with the TQ-dependent normalization of chronic accumulation of malonyl-CoA, elevation of acetyl-CoA carboxylase (ACC), fatty acid synthase, and fatty acid-binding proteins following chronic glucose overload. Together, these data suggest that TQ modulates the β-cell redox circuitry and enhances the sensitivity of β-cell metabolic pathways to glucose and GSIS under normal conditions as well as under hyperglycemia. This action is associated with the ability of TQ to regulate carbohydrate-to-lipid flux via downregulation of ACC and malonyl-CoA. PMID:26786775

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

PubMed

Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

2015-12-28

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

PubMed Central

Mir, Riyaz A.; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A.; Ammons, Shalis A.; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B.; Qiu, Fang; Band, Hamid

2015-01-01

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. PMID:26711270

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

PubMed

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

PubMed Central

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

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

Zhang, Jingjing; Shkrob, Ilya A.; Assary, Rajeev S.

We demonstrate that 9,10-Bis(2-methoxyethoxy)-1,2,3,4,5,6,7,8-octahydro-1,4:5,8-dimethanoanthracene redox shuttle molecule survives over 120 cycles with 100% overcharge ratio at C/5 rate in litium-ion batteries. Equally remarkably, in the presence of this electrolyte additive, the cell impedance becomes significantly lower compared to the control cells without this additive during the formation, normal cycling, and even under overcharge conditions.

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.

Mechanisms by which HPV Induces a Replication Competent Environment in Differentiating Keratinocytes

PubMed Central

Moody, Cary A.

2017-01-01

Human papillomaviruses (HPV) are the causative agents of cervical cancer and are also associated with other genital malignancies, as well as an increasing number of head and neck cancers. HPVs have evolved their life cycle to contend with the different cell states found in the stratified epithelium. Initial infection and viral genome maintenance occurs in the proliferating basal cells of the stratified epithelium, where cellular replication machinery is abundant. However, the productive phase of the viral life cycle, including productive replication, late gene expression and virion production, occurs upon epithelial differentiation, in cells that normally exit the cell cycle. This review outlines how HPV interfaces with specific cellular signaling pathways and factors to provide a replication-competent environment in differentiating cells. PMID:28925973

p21, an important mediator of quiescence during pituitary tumor formation, is dispensable for normal pituitary development during embryogenesis.

PubMed Central

Monahan, Pamela; Himes, Ashley D.; Parfieniuk, Agata; Raetzman, Lori T.

2011-01-01

A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components. PMID:22154697

Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells-update 2

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in low earth orbit (LEO) cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40 000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. This test was conducted at Hughes Aircraft Company under a NASA Lewis contract. The purpose was to investigate the effect of KOH concentration on cycle life. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The boiler plate test results are in the process of being validated using flight hardware and real time LEO test at the Naval Weapons Support Center (NWSC), Crane, Indiana under a NASA Lewis Contract. Six 48 Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16600 cycles during the continuing test.

Modelling of Fiber/Matrix Debonding of Composites Under Cyclic Loading

NASA Technical Reports Server (NTRS)

Naghipour, Paria; Pineda, Evan J.; Bednarcyk, Brett A.; Arnold, Steven M.

2013-01-01

The micromechanics theory, generalized method of cells (GMC), was employed to simulate the debonding of fiber/matrix interfaces, within a repeating unit cell subjected to global, cyclic loading, utilizing a cyclic crack growth law. Cycle dependent, interfacial debonding was implemented as a new module to the available GMC formulation. The degradation of interfacial stresses, with applied load cycles, was achieved via progressive evolution of the interfacial compliance. A periodic repeating unit cell, representing the fiber/matrix architecture of a composite, was subjected to combined normal and shear loadings, and degradation of the global transverse stress in successive cycles was monitored. The obtained results were compared to values from a corresponding finite element model. Reasonable agreement was achieved for combined normal and shear loading conditions, with minimal variation for pure loading cases. The local effects of interfacial debonding, and fatigue damage will later be combined as sub-models to predict the experimentally obtained fatigue life of Ti-15-3/Sic composites at the laminate level.

Coordination of the cell cycle is an important determinant of the syndrome of acute renal failure.

PubMed

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

2002-10-01

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

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

Proliferative status of primitive hematopoietic progenitors from patients with acute myelogenous leukemia (AML).

PubMed

Guan, Y; Hogge, D E

2000-12-01

One possible explanation for the competitive advantage that malignant cells in patients with acute myelogenous leukemia (AML) appear to have over normal hematopoietic elements is that leukemic progenitors proliferate more rapidly than their normal progenitor cell counterparts. To test this hypothesis, an overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in both colony-forming cell (CFC) and long-term culture initiating cell (LTC-IC) assays from the peripheral blood of nine patients with newly diagnosed AML. Culture of AML cells in serum-free medium with 100 ng/ml Steel factor (SF), 20 ng/ml interleukin 3 (IL-3) and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) for 16-24 h maintained the number of AML-CFC and LTC-IC at near input values (mean % input +/- s.d. for CFC and LTC-IC were 78 +/- 33 and 126 +/- 53, respectively). The addition of 20 muCi/ml high specific activity 3H-Tdr to these cultures reduced the numbers of both progenitor cell types from most of the patient samples substantially: mean % kill +/- s.d. for AML-CFC and LTC-IC were 64 +/- 27 and 82 +/- 16, respectively, indicating that a large proportion of both progenitor populations were actively cycling. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC were actively cycling (mean % kill +/- s.d.: 68 +/- 26 and 85 +/- 13, respectively). Interestingly, in six patient samples where a significant number of cytogenetically normal LTC-ICs were detected, the % kill of these cells (74 +/- 20) was similar to that of the abnormal progenitors. These data contrast with the predominantly quiescent cell cycle status of CFC and LTC-IC previously observed in steady-state peripheral blood from normal individuals but also provide evidence that a significant proportion of primitive malignant progenitors from AML patients are quiescent and therefore may be resistant to standard chemotherapeutic regimens.

In Vitro Alterations Do Not Reflect a Requirement for Host Cell Cycle Progression during Plasmodium Liver Stage Infection

PubMed Central

Hanson, Kirsten K.; March, Sandra; Ng, Shengyong; Bhatia, Sangeeta N.

2014-01-01

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection. PMID:25416236

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis

PubMed Central

Wu, Yuhsin V.; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O’Connor, Rachael; Geha, Rula C.; Somberg, C. Joy; Antonescu, Cristina; Singer, Samuel

2012-01-01

Well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n=84), WDLS (n=79), and normal fat (n=23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS compared to both WDLS and normal fat (15.2 fold and 27.8 fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for dedifferentiated liposarcomas. PMID:22170698

Differential Expression of Programmed Cell Death on the Follicular Development in Normal and Miniature Pig Ovary

PubMed Central

Kim, Sang Hwan; Min, Kwan Sik; Kim, Nam Hyung; Yoon, Jong Taek

2012-01-01

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs). PMID:23056260

Modulation of gene expression and cell-cycle signaling pathways by the EGFR inhibitor gefitinib (Iressa) in rat urinary bladder cancer.

PubMed

Lu, Yan; Liu, Pengyuan; Van den Bergh, Francoise; Zellmer, Victoria; James, Michael; Wen, Weidong; Grubbs, Clinton J; Lubet, Ronald A; You, Ming

2012-02-01

The epidermal growth factor receptor inhibitor Iressa has shown strong preventive efficacy in the N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) model of bladder cancer in the rat. To explore its antitumor mechanism, we implemented a systems biology approach to characterize gene expression and signaling pathways in rat urinary bladder cancers treated with Iressa. Eleven bladder tumors from control rats, seven tumors from rats treated with Iressa, and seven normal bladder epithelia were profiled by the Affymetrix Rat Exon 1.0 ST Arrays. We identified 713 downregulated and 641 upregulated genes in comparing bladder tumors versus normal bladder epithelia. In addition, 178 genes were downregulated and 96 genes were upregulated when comparing control tumors versus Iressa-treated tumors. Two coexpression modules that were significantly correlated with tumor status and treatment status were identified [r = 0.70, P = 2.80 × 10(-15) (bladder tumor vs. normal bladder epithelium) and r = 0.63, P = 2.00 × 10(-42) (Iressa-treated tumor vs. control tumor), respectively]. Both tumor module and treatment module were enriched for genes involved in cell-cycle processes. Twenty-four and twenty-one highly connected hub genes likely to be key drivers in cell cycle were identified in the tumor module and treatment module, respectively. Analysis of microRNA genes on the array chips showed that tumor module and treatment module were significantly associated with expression levels of let-7c (r = 0.54, P = 3.70 × 10(-8) and r = 0.73, P = 1.50 × 10(-65), respectively). These results suggest that let-7c downregulation and its regulated cell-cycle pathway may play an integral role in governing bladder tumor suppression or collaborative oncogenesis and that Iressa exhibits its preventive efficacy on bladder tumorigenesis by upregulating let-7 and inhibiting the cell cycle. Cell culture study confirmed that the increased expression of let-7c decreases Iressa-treated bladder tumor cell growth. The identified hub genes may also serve as pharmacodynamic or efficacy biomarkers in clinical trials of chemoprevention in human bladder cancer. ©2011 AACR.

Scalloped and Yorkie are required for cell cycle re-entry of quiescent cells after tissue damage.

PubMed

Meserve, Joy H; Duronio, Robert J

2015-08-15

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. We used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. We discovered that Hippo signaling regulates compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage. © 2015. Published by The Company of Biologists Ltd.

Primary fibroblasts from BRCA1 heterozygotes display an abnormal G1/S cell cycle checkpoint following UVA irradiation but show normal levels of micronuclei following oxidative stress or mitomycin C treatment.

PubMed

Shorrocks, Julie; Tobi, Simon E; Latham, Harry; Peacock, John H; Eeles, Ros; Eccles, Diana; McMillan, Trevor J

2004-02-01

There is evidence to suggest that the breast cancer predisposing gene, BRCA1, is involved in cell cycle control and the response to damage but mouse brca1+/- heterozygotes have no distinctive phenotype. Here the response to the three forms of cellular stress was examined in primary human fibroblasts from individuals with a +/+ or +/- genotype for BRCA1. Fibroblasts from individuals carrying mutations in the BRCA1 gene were compared with those from those wild-type for BRCA1 in their response to long wavelength uv (UVA), hydrogen peroxide, and mitomycin C (MMC). Cell cycle progression and micronucleus formation (MN) were used as end points. After UVA treatment there was no difference between +/- and +/+ cells in the initial fall in DNA synthetic activity (G(1) arrest) but the reentry into S-phase was restored at a faster rate in the BRCA1+/- cells after UVA exposure. Thus, for three normal (+/+) cell lines irradiated in monolayer, S-phase values averaged 15 +/- 3.7% 14 h post-UVA (1 x 10(5) J/m(2)), as compared with 35.7 +/- 1.9 (range) for two BRCA1(+/-) strains. Because a defective G(1)/S checkpoint in BRCA1 heterozygotes could lead to a greater proportion of S-phase cells with unrepaired DNA damage (strand breaks) and a resultant increase in chromosomal instability, the frequency of micronuclei induced by UVA was examined. Three normal (+/+) and three mutant (+/-) strains (two of which were used in the cell cycle experiments) produced mean micronuclei frequencies of 0.077 +/- 0.016 and 0.094 +/- 0.04/binucleate cell respectively (not statistically significant), 48 h after UVA exposure. No differences were found between BRCA1+/+ and +/- cells in MN formation after treatment with MMC or hydrogen peroxide. Our data suggest a defective G(1)/S checkpoint in cells from BRCA1 heterozygotes in response to UVA although this is not reflected in genomic instability as measured by micronuclei induction after oxidative stress or MMC treatment.

Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response

PubMed Central

dos Santos, Nathália Villa; Matias, Andreza Cândido; Higa, Guilherme Shigueto Vilar; Kihara, Alexandre Hiroaki; Cerchiaro, Giselle

2015-01-01

The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion. PMID:26583055

KPU-300, a Novel Benzophenone–Diketopiperazine–Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase

PubMed Central

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300. PMID:26716455

KPU-300, a Novel Benzophenone-Diketopiperazine-Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase.

PubMed

Okuyama, Kohei; Kaida, Atsushi; Hayashi, Yoshiki; Hayashi, Yoshio; Harada, Kiyoshi; Miura, Masahiko

2015-01-01

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

PubMed

Davidson, E J; Morris, L S; Scott, I S; Rushbrook, S M; Bird, K; Laskey, R A; Wilson, G E; Kitchener, H C; Coleman, N; Stern, P L

2003-01-27

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

[Dose rate-dependent cellular and molecular effects of ionizing radiation].

PubMed

Przybyszewski, Waldemar M; Wideł, Maria; Szurko, Agnieszka; Maniakowski, Zbigniew

2008-09-11

The aim of radiation therapy is to kill tumor cells while minimizing damage to normal cells. The ultimate effect of radiation can be apoptotic or necrotic cell death as well as cytogenetic damage resulting in genetic instability and/or cell death. The destructive effects of radiation arise from direct and indirect ionization events leading to peroxidation of macromolecules, especially those present in lipid-rich membrane structures as well as chromatin lipids. Lipid peroxidative end-products may damage DNA and proteins. A characteristic feature of radiation-induced peroxidation is an inverse dose-rate effect (IDRE), defined as an increase in the degree of oxidation(at constant absorbed dose) accompanying a lower dose rate. On the other hand, a low dose rate can lead to the accumulation of cells in G2, the radiosensitive phase of the cell cycle since cell cycle control points are not sensitive to low dose rates. Radiation dose rate may potentially be the main factor improving radiotherapy efficacy as well as affecting the intensity of normal tissue and whole-body side effects. A better understanding of dose rate-dependent biological effects may lead to improved therapeutic intervention and limit normal tissue reaction. The study reviews basic biological effects that depend on the dose rate of ionizing radiation.

Genome-wide screen identifies a novel prognostic signature for breast cancer survival

DOE PAGES

Mao, Xuan Y.; Lee, Matthew J.; Zhu, Jeffrey; ...

2017-01-21

Large genomic datasets in combination with clinical data can be used as an unbiased tool to identify genes important in patient survival and discover potential therapeutic targets. We used a genome-wide screen to identify 587 genes significantly and robustly deregulated across four independent breast cancer (BC) datasets compared to normal breast tissue. Gene expression of 381 genes was significantly associated with relapse-free survival (RFS) in BC patients. We used a gene co-expression network approach to visualize the genetic architecture in normal breast and BCs. In normal breast tissue, co-expression cliques were identified enriched for cell cycle, gene transcription, cell adhesion,more » cytoskeletal organization and metabolism. In contrast, in BC, only two major co-expression cliques were identified enriched for cell cycle-related processes or blood vessel development, cell adhesion and mammary gland development processes. Interestingly, gene expression levels of 7 genes were found to be negatively correlated with many cell cycle related genes, highlighting these genes as potential tumor suppressors and novel therapeutic targets. A forward-conditional Cox regression analysis was used to identify a 12-gene signature associated with RFS. A prognostic scoring system was created based on the 12-gene signature. This scoring system robustly predicted BC patient RFS in 60 sampling test sets and was further validated in TCGA and METABRIC BC data. Our integrated study identified a 12-gene prognostic signature that could guide adjuvant therapy for BC patients and includes novel potential molecular targets for therapy.« less

Genome-wide screen identifies a novel prognostic signature for breast cancer survival

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

Mao, Xuan Y.; Lee, Matthew J.; Zhu, Jeffrey

Large genomic datasets in combination with clinical data can be used as an unbiased tool to identify genes important in patient survival and discover potential therapeutic targets. We used a genome-wide screen to identify 587 genes significantly and robustly deregulated across four independent breast cancer (BC) datasets compared to normal breast tissue. Gene expression of 381 genes was significantly associated with relapse-free survival (RFS) in BC patients. We used a gene co-expression network approach to visualize the genetic architecture in normal breast and BCs. In normal breast tissue, co-expression cliques were identified enriched for cell cycle, gene transcription, cell adhesion,more » cytoskeletal organization and metabolism. In contrast, in BC, only two major co-expression cliques were identified enriched for cell cycle-related processes or blood vessel development, cell adhesion and mammary gland development processes. Interestingly, gene expression levels of 7 genes were found to be negatively correlated with many cell cycle related genes, highlighting these genes as potential tumor suppressors and novel therapeutic targets. A forward-conditional Cox regression analysis was used to identify a 12-gene signature associated with RFS. A prognostic scoring system was created based on the 12-gene signature. This scoring system robustly predicted BC patient RFS in 60 sampling test sets and was further validated in TCGA and METABRIC BC data. Our integrated study identified a 12-gene prognostic signature that could guide adjuvant therapy for BC patients and includes novel potential molecular targets for therapy.« less

Cardiac Myocyte Cell Cycle Control in Development, Disease and Regeneration

PubMed Central

Ahuja, Preeti; Sdek, Patima; Maclellan, W. Robb

2009-01-01

Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle soon after birth in mammals. Although the extent to which adult cardiac myocytes are capable of cell cycle reentry is controversial and species-specific differences may exist, it appears that for the vast majority of adult cardiac myocytes the predominant form of growth postnatally is an increase in cell size (hypertrophy) not number. Unfortunately, this limits the ability of the heart to restore function after any significant injury. Interst in novel regenerative therapies has led to the accumulation of much information on the mechanisms that regulate the rapid proliferation of cardiac myocytes in utero, their cell cycle exit in the perinatal period and the permanent arrest (terminal differentiation) in adult myocytes. The recent identification of cardiac progenitor cells capable of giving rise to cardiac myocyte-like cells has challenged the dogma that the heart is a terminally differentiated organ and opened new prospects for cardiac regeneration. In this review, we summarize the current understanding of cardiomyocyte cell cycle control in normal development and disease. In addition, we also discuss the potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration. PMID:17429040

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

INITIATION OF MITOSIS IN RELATION TO THE CELL CYCLE FOLLOWING FEEDING OF STARVED CHICKENS

PubMed Central

Cameron, Ivan L.; Cleffmann, Günter

1964-01-01

Cellular proliferation of newly hatched chickens was depressed by starving them for 2.5 to 3.5 days. Starvation may hold proliferative cells in different parts of the cell cycle. In order to find where in the cell cycle these cells are held, the animals were fed and the following events were measured as a function of time after the start of feeding: (1) the mitotic index, and (2) the DNA synthetic index (number of cells in DNA synthesis 1 hour after injection of H3-thymidine). The duration of the cell's DNA synthetic period (S) was measured, permitting a more exact description of the cell cycle. Analysis of the duodenal and esophageal epithelia shows that feeding initiates cell division by stimulating cells from the G1 part of the mitotic cycle in the duodenum. In the esophagus some of the cells were either stopped or slowed down in G1, and another group of cells in G2. Feeding simultaneously stimulates both cell groups; the former moves into S, the latter into mitosis. The S period in starved animals is a little longer than that in normally fed animals but the extension can be attributed to a slightly decreased body temperature. PMID:14153479

B-vitamin deficiency is protective in experimental colitis

USDA-ARS?s Scientific Manuscript database

Methionine (Met) cycle activity is critical for normal cell functions and requires B-vitamin (B6/B12) as cofactors. Sadenosylhomocysteine (SAH) is a Met cycle intermediates that is known to inhibit methyltransferases. Met metabolism is altered in patients with inflammatory bowel disease (IBD), but M...

Anti-inflammatory properties of methylthioadenosine in experimental colitis

USDA-ARS?s Scientific Manuscript database

The methionine (Met) metabolic cycle is critical for normal cell functions. Met cycle disruption has been implicated in disease, such as alcoholic liver disease (ALD) and multiple sclerosis (MS). Studies in animal models of ALD and MS have shown that the Met metabolite methylthioadenosine (MTA) has ...

Granulosa cell cycle regulation and steroidogenesis in a high androstenedione follicular microenvironment

USDA-ARS?s Scientific Manuscript database

Anovulatory infertility (either chronic or sporadic anovulation) affects up to 40% of infertile women. In fact, sporadic anovulation in humans may often go undetected. Recent literature has reported that 8-13% of normally menstruating women (250 total, two reproductive cycles) exhibit sporadic anovu...

Induction of apoptosis and cell cycle arrest in L-1210 murine lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one.

PubMed

Pedrini, Fernanda Spezia; Chiaradia, Louise Domeneghini; Licínio, Marley Aparecida; de Moraes, Ana Carolina Rabello; Curta, Juliana Costa; Costa, Aline; Mascarello, Alessandra; Creczinsky-Pasa, Tânia Beatriz; Nunes, Ricardo José; Yunes, Rosendo Augusto; Santos-Silva, Maria Cláudia

2010-09-01

New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic leukemia cells L-1210. A series of ten chalcones derived from 2-naphtaldehyde and corresponding acetophenones were prepared by aldolic condensation, using methanol as solvent under basic conditions, at room temperature for 24 h. The cell viability was determined by MTT colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after propidium iodide staining. The apoptosis induction was assessed by exposure to phosphatidylserine (ANNEXIN V-FITC). Cytometric analysis was performed to evaluate the expression of p53, Bcl-2 and Bax protein. The caspase-3 expression was studied by immunoblotting analysis. A preliminary screening of a series of ten chalcones derived from 2-naphtaldehyde showed that chalcone 8, (2E)-3-(2-naphtyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one, had the highest cytotoxic effect (IC50 of 54 microM), but not in normal human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8, its effect on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in Bax:Bcl-2 ratio and increased p53 expression and caspase-3 activation. The studied chalcone 8 has cytotoxic effect against L-1210 lymphoblastic leukaemic cells, and this effect is associated with increase of p-53 and Bax expression.

Determination of cell cycle phases in live B16 melanoma cells using IRMS.

PubMed

Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

2013-07-21

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.

PubMed

Massodi, Iqbal; Moktan, Shama; Rawat, Aruna; Bidwell, Gene L; Raucher, Drazen

2010-01-15

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

Colon Cancer-Upregulated Long Non-Coding RNA lincDUSP Regulates Cell Cycle Genes and Potentiates Resistance to Apoptosis.

PubMed

Forrest, Megan E; Saiakhova, Alina; Beard, Lydia; Buchner, David A; Scacheri, Peter C; LaFramboise, Thomas; Markowitz, Sanford; Khalil, Ahmad M

2018-05-09

Long non-coding RNAs (lncRNAs) are frequently dysregulated in many human cancers. We sought to identify candidate oncogenic lncRNAs in human colon tumors by utilizing RNA sequencing data from 22 colon tumors and 22 adjacent normal colon samples from The Cancer Genome Atlas (TCGA). The analysis led to the identification of ~200 differentially expressed lncRNAs. Validation in an independent cohort of normal colon and patient-derived colon cancer cell lines identified a novel lncRNA, lincDUSP, as a potential candidate oncogene. Knockdown of lincDUSP in patient-derived colon tumor cell lines resulted in significantly decreased cell proliferation and clonogenic potential, and increased susceptibility to apoptosis. The knockdown of lincDUSP affects the expression of ~800 genes, and NCI pathway analysis showed enrichment of DNA damage response and cell cycle control pathways. Further, identification of lincDUSP chromatin occupancy sites by ChIRP-Seq demonstrated association with genes involved in the replication-associated DNA damage response and cell cycle control. Consistent with these findings, lincDUSP knockdown in colon tumor cell lines increased both the accumulation of cells in early S-phase and γH2AX foci formation, indicating increased DNA damage response induction. Taken together, these results demonstrate a key role of lincDUSP in the regulation of important pathways in colon cancer.

Dietary glutamate reduces systemic but not intestinal leucine oxidation in protein malnourished piglets

USDA-ARS?s Scientific Manuscript database

The methionine (Met) metabolic cycle is critical for normal cell functions. Met cycle disruption has been implicated in disease, such as alcoholic liver disease (ALD) and multiple sclerosis (MS). Studies in animal models of ALD and MS have shown that the Met metabolite methylthioadenosine (MTA) has ...

HEAT SHOCK FACTOR 1-MEDIATED THERMOTOLERANCE PREVENTS CELL DEATH AND RESULTS IN G2/M CELL CYCLE ARREST

EPA Science Inventory

Mammalian cells respond to stress by activating heat shock transcription factors (e.g., HSF1) that regulate increased synthesis of heat shock proteins (HSPs). HSPs mediate protection from deleterious effects of stress by preventing permanent disruption of normal cellular mitosis...

Study of the circadian rhythm in radiation response

USDA-ARS?s Scientific Manuscript database

Gamma-Radiation is often used for the treatment of solid tumors. It induces DNA double-stranded breaks that lead to cell cycle arrest or apoptosis of tumor cells. However, such treatment could also damage normal host tissues that need cell proliferation for function. We have reported previously that...

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

PubMed

Cen, Yan-Hui; Guo, Wen-Wen; Luo, Bin; Lin, Yong-Da; Zhang, Qing-Mei; Zhou, Su-Fang; Luo, Guo-Rong; Xiao, Shao-Wen; Xie, Xiao-Xun

2012-10-01

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

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

Tian, Junqiang; Doi, Hiroshi; Saar, Matthias

2013-12-01

Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome.more » The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.« less

The up-regulation of miR-300 in gastric cancer and its effects on cells malignancy

PubMed Central

Shen, Zhen; Li, Chunsheng; Zhang, Kai; Yu, Wei; Xiao, Huijie; Li, Bo; Liu, Tongjun

2015-01-01

Objective: In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of gastric cancer cells. Methods: MicroRNA and protein expression patterns were compared between gastric cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in AGS gastric cancer cells. Results: We observed that miR-300 expression was frequently and dramatically up-regulated in human gastric cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote gastric cancer cell proliferation and invasion by increasing p53 expression. Conclusion: Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in gastric cancer cell proliferation during gastric tumorigenesis. PMID:26221215

The up-regulation of miR-300 in gastric cancer and its effects on cells malignancy.

PubMed

Shen, Zhen; Li, Chunsheng; Zhang, Kai; Yu, Wei; Xiao, Huijie; Li, Bo; Liu, Tongjun

2015-01-01

In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of gastric cancer cells. MicroRNA and protein expression patterns were compared between gastric cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in AGS gastric cancer cells. We observed that miR-300 expression was frequently and dramatically up-regulated in human gastric cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote gastric cancer cell proliferation and invasion by increasing p53 expression. Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in gastric cancer cell proliferation during gastric tumorigenesis.

Redistribution of cell cycle by arsenic trioxide is associated with demethylation and expression changes of cell cycle related genes in acute promyelocytic leukemia cell line (NB4).

PubMed

Hassani, Saeed; Khaleghian, Ali; Ahmadian, Shahin; Alizadeh, Shaban; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2018-01-01

PML-RARα perturbs the normal epigenetic setting, which is essential to oncogenic transformation in acute promyelocytic leukemia (APL). Transcription induction and recruitment of DNA methyltransferases (DNMTs) by PML-RARα and subsequent hypermethylation are components of this perturbation. Arsenic trioxide (ATO), an important drug in APL therapy, concurrent with degradation of PML-RARα induces cell cycle change and apoptosis. How ATO causes cell cycle alteration has remained largely unexplained. Here, we investigated DNA methylation patterns of cell cycle regulatory genes promoters, the effects of ATO on the methylated genes and cell cycle distribution in an APL cell line, NB4. Analysis of promoter methylation status of 22 cell cycle related genes in NB4 revealed that CCND1, CCNE1, CCNF, CDKN1A, GADD45α, and RBL1 genes were methylated 60.7, 84.6, 58.6, 8.7, 33.4, and 73.7%, respectively, that after treatment with 2 μM ATO for 48 h, turn into 0.6, 13.8, 0.1, 6.6, 10.7, and 54.5% methylated. ATO significantly reduced the expression of DNMT1, 3A, and 3B. ATO induced the expression of CCND1, CCNE1, and GADD45α genes, suppressed the expression of CCNF and CDKN1A genes, which were consistent with decreased number of cells in G1 and S phases and increased number of cells in G2/M phase. In conclusion, demethylation and alteration in the expression level of the cell cycle related genes may be possible mechanisms in ATO-induced cell cycle arrest in APL cells. It may suggest that ATO by demethylation of CCND1 and CCNE1 and their transcriptional activation accelerates G1 and S transition into the G2/M cell cycle arrest.

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

PubMed

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

2003-07-15

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

Radioprotection: smart games with death.

PubMed

Gudkov, Andrei V; Komarova, Elena A

2010-07-01

The efficacy of cancer treatment by radiation and chemotherapeutic drugs is often limited by severe side effects that primarily affect the hematopoietic system and the epithelium of the gastrointestinal tract. Progress in understanding differences in the mechanisms involved in the responses of normal and tumor cells to genotoxic stress has led to the development of new rational approaches to selective protection of normal cells, such as suppression of apoptosis by pharmacological inhibition of p53 or activation of NF-kappaB. Another promising approach presented in this issue by Johnson et al. is based on the idea of using pharmacological inhibitors of cyclin-dependent kinases (CDKs) to convert normal cells into a radioresistant state by inducing reversible cell cycle arrest at the G1/S transition. The evidence indicates that this approach is likely to be specific for protection of normal cells and may, therefore, have clinical potential as an adjuvant in anticancer therapies.

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis.

PubMed

Wu, Yuhsin V; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O'Connor, Rachael; Geha, Rula C; Joy Somberg, C; Antonescu, Cristina; Singer, Samuel

2012-04-01

Well-differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n = 84), WDLS (n = 79), and normal fat (n = 23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS when compared to both WDLS and normal fat (15.2- and 27.8-fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBPα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for DDLS. Copyright © 2011 Wiley Periodicals, Inc.

MiR-300 regulate the malignancy of breast cancer by targeting p53.

PubMed

Xu, Xiao-Heng; Li, Da-Wei; Feng, Hui; Chen, Hong-Mei; Song, Yan-Qiu

2015-01-01

In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of breast cancer (BC) cells. MicroRNA and protein expression patterns were compared between breast cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in MCF-7 breast cancer cells. We observed that miR-300 expression was frequently and dramatically up-regulated in human breast cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote breast cancer cell proliferation and invasion by regulating p53 expression. Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in breast cancer cell proliferation during breast tumorigenesis.

MiR-300 regulate the malignancy of breast cancer by targeting p53

PubMed Central

Xu, Xiao-Heng; Li, Da-Wei; Feng, Hui; Chen, Hong-Mei; Song, Yan-Qiu

2015-01-01

Objective: In this study, we investigated the role of miR-300 in regulating cell proliferation and invasion of breast cancer (BC) cells. Methods: MicroRNA and protein expression patterns were compared between breast cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of miR-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in MCF-7 breast cancer cells. Results: We observed that miR-300 expression was frequently and dramatically up-regulated in human breast cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of miR-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of miR-300, suggesting that miR-300 might promote breast cancer cell proliferation and invasion by regulating p53 expression. Conclusion: Our findings indicated that miR-300 up-regulation might exert some sort of antagonistic function by targeting p53 in breast cancer cell proliferation during breast tumorigenesis. PMID:26221232

Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

PubMed Central

Mi, Da; Carr, Catherine B.; Georgala, Petrina A.; Huang, Yu-Ting; Manuel, Martine N.; Jeanes, Emily; Niisato, Emi; Sansom, Stephen N.; Livesey, Frederick J.; Theil, Thomas; Hasenpusch-Theil, Kerstin; Simpson, T. Ian; Mason, John O.; Price, David J.

2013-01-01

Summary The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct. PMID:23622063

The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

PubMed Central

Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

2013-01-01

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

Topoisomerase II Inhibitors and Poisons, and the Influence of Cell Cycle Checkpoints.

PubMed

D Arcy, Nicholas; Gabrielli, Brian

2017-01-01

Interactions between the decatenation checkpoint and Topoisomerase II (TopoII) are vital for maintaining integrity of the genome. Agents that target this enzyme have been in clinical use in cancer therapy for over 30 years with great success. The types of compounds that have been developed to target TopoII are broadly divided into poisons and catalytic inhibitors. The TopoII poisons are in clinical use as anti-cancer therapies, although in common to most chemotherapeutic agents, they display considerable normal tissue toxicity. Inhibition of the TopoIIb isoform has been implicated in this cytotoxicity. Response to TopoII active agents is determined by several factors, but cell cycle checkpoints play a large role in sensitivity and resistance. The G2/M phase checkpoints are of particular importance in considering the effectiveness of these drugs and are reviewed in this article. Functionality of the ATM dependent decatenation checkpoint may represent a new avenue for selective cancer therapy. Here we review the function of TopoII, the anti-cancer mechanisms and limitations of current catalytic inhibitors and poisons, and their influence on cell cycle checkpoints. We will also assess potential new mechanisms for targeting this enzyme to limit normal tissue toxicity, and how the cell cycle checkpoint triggered by these drugs may provide an alternative and possibly better target for novel therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

ULTRASTRUCTURE, STEROIDOGENIC POTENTIAL, AND ENERGY METABOLISM OF THE SNELL ADRENOCORTICAL CARCINOMA 494

PubMed Central

Kimmel, G. L.; Péron, F. G.; Haksar, A.; Bedigian, E.; Robidoux, W. F.; Lin, M. T.

1974-01-01

Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11β-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11β-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact. PMID:4366105

Aberrant light directly impairs mood and learning through melanopsin-expressing neurons.

PubMed

LeGates, Tara A; Altimus, Cara M; Wang, Hui; Lee, Hey-Kyoung; Yang, Sunggu; Zhao, Haiqing; Kirkwood, Alfredo; Weber, E Todd; Hattar, Samer

2012-11-22

The daily solar cycle allows organisms to synchronize their circadian rhythms and sleep-wake cycles to the correct temporal niche. Changes in day-length, shift-work, and transmeridian travel lead to mood alterations and cognitive function deficits. Sleep deprivation and circadian disruption underlie mood and cognitive disorders associated with irregular light schedules. Whether irregular light schedules directly affect mood and cognitive functions in the context of normal sleep and circadian rhythms remains unclear. Here we show, using an aberrant light cycle that neither changes the amount and architecture of sleep nor causes changes in the circadian timing system, that light directly regulates mood-related behaviours and cognitive functions in mice. Animals exposed to the aberrant light cycle maintain daily corticosterone rhythms, but the overall levels of corticosterone are increased. Despite normal circadian and sleep structures, these animals show increased depression-like behaviours and impaired hippocampal long-term potentiation and learning. Administration of the antidepressant drugs fluoxetine or desipramine restores learning in mice exposed to the aberrant light cycle, suggesting that the mood deficit precedes the learning impairments. To determine the retinal circuits underlying this impairment of mood and learning, we examined the behavioural consequences of this light cycle in animals that lack intrinsically photosensitive retinal ganglion cells. In these animals, the aberrant light cycle does not impair mood and learning, despite the presence of the conventional retinal ganglion cells and the ability of these animals to detect light for image formation. These findings demonstrate the ability of light to influence cognitive and mood functions directly through intrinsically photosensitive retinal ganglion cells.

Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein.

PubMed

Sheldon, Lynn A

2017-01-01

The regulation of cell cycle progression by steroid hormones and growth factors is important for maintaining normal cellular processes including development and cell proliferation. Deregulated progression through the G1/S and G2/M cell cycle transitions can lead to uncontrolled cell proliferation and cancer. The transcription factor E2F1, a key cell cycle regulator, targets genes encoding proteins that regulate cell cycle progression through the G1/S transition as well as proteins important in DNA repair and apoptosis. E2F1 expression and activity is inhibited by inorganic arsenic (iAs) that has a dual role as a cancer therapeutic and as a toxin that leads to diseases including cancer. An understanding of what underlies this dichotomy will contribute to understanding how to use iAs as a more effective therapeutic and also how to treat cancers that iAs promotes. Here, we show that quiescent breast adenocarcinoma MCF-7 cells treated with 17-β estradiol (E2) progress through the cell cycle, but few cells treated with E2 + iAs progress from G1 into S-phase due to a block in cell cycle progression. Our data support a model in which iAs inhibits the dissociation of E2F1 from the tumor suppressor, retinoblastoma protein (pRB) due to changes in pRB phosphorylation which leads to decreased E2F1 transcriptional activity. These findings present an explanation for how iAs can disrupt cell cycle progression through E2F1-pRB and has implications for how iAs acts as a cancer therapeutic as well as how it may promote tumorigenesis through decreased DNA repair.

Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision

PubMed Central

Phua, Siew Cheng; Chiba, Shuhei; Suzuki, Masako; Su, Emily; Roberson, Elle C.; Pusapati, Ganesh V.; Setou, Mitsutoshi; Rohatgi, Rajat; Reiter, Jeremy F.; Ikegami, Koji; Inoue, Takanari

2017-01-01

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate to distal cilia. This triggers otherwise forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. Whilst cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer. PMID:28086093

Evidence against involvement of the acid lysosomal sphingomyelinase in the tumor-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts.

PubMed Central

Andrieu, N; Salvayre, R; Levade, T

1994-01-01

The hydrolysis of sphingomyelin (SPM) has been reported to mediate a number of responses to extracellular agents, including cytokines. The so-called SPM cycle may result from the activation of different types of sphingomyelinases (SPMases). We investigated the hypothetical contribution of acid lysosomal SPMase in the SPM signal-transduction pathway. We examined the ability of human skin fibroblasts with a genetic deficiency of acid lysosomal SPMase activity to respond to tumour necrosis factor alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). We report that both cytokines promoted SPM hydrolysis in fibroblasts derived from patients with Niemann-Pick disease or I-cell disease, similar to that observed in normal cells. Treatment of normal fibroblasts with cationic amphiphilic drugs resulted in inhibition of acid SPMase activity, but had no effect on cytokine-induced SPM turnover. In addition, TNF-alpha and IL-1 beta stimulated [3H]thymidine incorporation in Niemann-Pick fibroblasts, as in normal cells. Thus our results argue against a role for acid endolysosomal SPMase in mediating the cytokine-induced SPM signalling cascade. Images Figure 2 PMID:7980390

Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines.

PubMed

Haddad, A Q; Venkateswaran, V; Viswanathan, L; Teahan, S J; Fleshner, N E; Klotz, L H

2006-01-01

Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,2'-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,2'-DHC and fisetin in PCa, and to establish their effectiveness in vivo.

The B-MYB Transcriptional Network Guides Cell Cycle Progression and Fate Decisions to Sustain Self-Renewal and the Identity of Pluripotent Stem Cells

PubMed Central

Zhan, Ming; Riordon, Daniel R.; Yan, Bin; Tarasova, Yelena S.; Bruweleit, Sarah; Tarasov, Kirill V.; Li, Ronald A.; Wersto, Robert P.; Boheler, Kenneth R.

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. PMID:22936984

The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

PubMed

Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (anticancer effects of 3-bromopyruvate through antagonizing Warburg effect).

PubMed

El Sayed, Salah Mohamed; Mahmoud, Ahmed Alamir; El Sawy, Samer Ahmed; Abdelaal, Esam Abdelrahim; Fouad, Amira Murad; Yousif, Reda Salah; Hashim, Marwa Shaban; Hemdan, Shima Badawy; Kadry, Zainab Mahmoud; Abdelmoaty, Mohamed Ahmed; Gabr, Adel Gomaa; Omran, Faten M; Nabo, Manal Mohamed Helmy; Ahmed, Nagwa Sayed

2013-11-01

Cancer cells undergo an increased steady-state ROS condition compared to normal cells. Among the major metabolic differences between cancer cells and normal cells is the dependence of cancer cells on glycolysis as a major source of energy even in the presence of oxygen (Warburg effect). In Warburg effect, glucose is catabolized to lactate that is extruded through monocarboxylate transporters to the microenvironment of cancer cells, while in normal cells, glucose is metabolized into pyruvate that is not extruded. Pyruvate is a potent antioxidant, while lactate has no antioxidant effect. Pyruvate in normal cells may be further metabolized to acetyl CoA and then through Krebs cycle with production of antioxidant intermediates e.g. citrate, malate and oxaloacetate together with the reducing equivalents (NADH.H+). Through activity of mitochondrial transhydrogenase, NADH.H+ replenishes NADPH.H+, coenzyme of glutathione reductase which replenishes reduced form of glutathione (potent antioxidant). This enhances antioxidant capacities of normal cells, while cancer cells exhibiting Warburg effect may be deprived of all that antioxidant capabilities due to loss of extruded lactate (substrate for Krebs cycle). Although intrinsic oxidative stress in cancer cells is high, it may be prevented from reaching progressively increasing levels that are cytotoxic to cancer cells. This may be due to some antioxidant effects exerted by hexokinase II (HK II) and NADPH.H+ produced through HMP shunt. Glycolytic phenotype in cancer cells maintains a high non-toxic oxidative stress in cancer cells and may be responsible for their malignant behavior. Through HK II, glycolysis fuels the energetic arm of malignancy, the mitotic arm of malignancy (DNA synthesis through HMP shunt pathway) and the metastatic arm of malignancy (hyaluronan synthesis through uronic acid pathway) in addition to the role of phosphohexose isomerase (autocrine motility factor). All those critical three arms start with the substrate G6P that is a direct product of HK II. 3-bromopyruvate (3BP, inhibitor of HK II) may prove as a promising anticancer and antimetastatic agent based on antagonizing the Warburg effect and disturbing the malignant behavior in cancer cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells.

PubMed

Ramljak, Danica; Romanczyk, Leo J; Metheny-Barlow, Linda J; Thompson, Nicole; Knezevic, Vladimir; Galperin, Mikhail; Ramesh, Arun; Dickson, Robert B

2005-04-01

A naturally occurring, cocoa-derived pentameric procyanidin (pentamer) was previously shown to cause G0/G1 cell cycle arrest in human breast cancer cells by an unknown molecular mechanism. Here, we show that pentamer selectively inhibits the proliferation of human breast cancer cells (MDA MB-231, MDA MB-436, MDA MB-468, SKBR-3, and MCF-7) and benzo(a)pyrene-immortalized 184A1N4 and 184B5 cells. In contrast, normal human mammary epithelial cells in primary culture and spontaneously immortalized MCF-10A cells were significantly resistant. We evaluated whether this differential response to pentamer may involve depolarization of the mitochondrial membrane. Pentamer caused significant depolarization of mitochondrial membrane in MDA MB231 cells but not the more normal MCF-10A cells, whereas other normal and tumor cell lines tested gave variable results. Further investigations, using a proteomics approach with pentamer-treated MDA MB-231, revealed a specific dephosphorylation, without changes in protein expression, of several G1-modulatory proteins: Cdc2 (at Tyr15), forkhead transcription factor (at Ser256, the Akt phosphorylation site) and p53 (Ser392). Dephosphorylation of p53 (at Ser392) by pentamer was confirmed in MDA MB-468 cells. However, both expression and phosphorylation of retinoblastoma protein were decreased after pentamer treatment. Our results show that breast cancer cells are selectively susceptible to the cytotoxic effects of pentameric procyanidin, and suggest that inhibition of cellular proliferation by this compound is associated with the site-specific dephosphorylation or down-regulation of several cell cycle regulatory proteins.

How does the Xenopus laevis embryonic cell cycle avoid spatial chaos?

PubMed Central

Gelens, Lendert; Huang, Kerwyn Casey; Ferrell, James E.

2015-01-01

Summary Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume, and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration, and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells. PMID:26212326

TP53 status and response to chemotherapy in breast cancer.

PubMed

Bertheau, Philippe; Espié, Marc; Turpin, Elisabeth; Lehmann, Jacqueline; Plassa, Louis-François; Varna, Mariana; Janin, Anne; de Thé, Hugues

2008-01-01

Despite its central role in the control of apoptosis, senescence and cell cycle arrest, the tumor suppressor protein p53 remains an enigma for its possible role in predicting response to chemotherapy in cancer patients. Many studies remained inconclusive, others showed a better response for tumors with normal p53, and some recent studies showed adverse effects of normal p53 for response to treatment. p53 is not only a powerful pro-apoptotic factor in response to drug-induced DNA damages but also a potential inducer of cell cycle arrest, protecting tumor cells from further cytotoxic damages. Our review describes the classical as well as the more recent concepts. In order to draw definite conclusions, future works should use more reliable methods to assess the TP53 status and should address more homogeneous tumor subpopulations treated with homogeneous chemotherapy regimens. Copyright 2008 S. Karger AG, Basel.

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

PubMed Central

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-01-01

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

PubMed

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-09-08

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Epidermal stem cells: location, potential and contribution to cancer.

PubMed

Ambler, C A; Määttä, A

2009-01-01

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells.

Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override

PubMed Central

Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

2014-01-01

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25. PMID:24666325

Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

PubMed

Lee, Wooram; Lazar, Iulia M

2014-07-01

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

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

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

PubMed Central

Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

2016-01-01

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting

PubMed Central

Lindeman, Geoffrey J.; Dagnino, Lina; Gaubatz, Stefan; Xu, Yuhui; Bronson, Roderick T.; Warren, Henry B.; Livingston, David M.

1998-01-01

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue. PMID:9553039

Lessons From the First Comprehensive Molecular Characterization of Cell Cycle Control in Rodent Insulinoma Cell Lines

PubMed Central

Cozar-Castellano, Irene; Harb, George; Selk, Karen; Takane, Karen; Vasavada, Rupangi; Sicari, Brian; Law, Brian; Zhang, Pili; Scott, Donald K.; Fiaschi-Taesch, Nathalie; Stewart, Andrew F.

2008-01-01

OBJECTIVE—Rodent insulinoma cell lines may serve as a model for designing continuously replicating human β-cell lines and provide clues as to the central cell cycle regulatory molecules in the β-cell. RESEARCH DESIGN AND METHODS—We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics. RESULTS—1) Despite their common T-antigen–derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat β-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate β-cell differentiation and function. CONCLUSIONS—These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human β-cells. PMID:18650366

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

PubMed Central

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

The Molecular Chaperone Hsp90 Is Required for Cell Cycle Exit in Drosophila melanogaster

PubMed Central

Bandura, Jennifer L.; Jiang, Huaqi; Nickerson, Derek W.; Edgar, Bruce A.

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

PubMed

Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

2013-01-01

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis.

Biological effects of radiation, metabolic and replication kinetics alterations

NASA Technical Reports Server (NTRS)

Post, J.

1972-01-01

The biological effects of radiation upon normal and cancerous tissues were studied. A macromolecular precursor of DNA, 3ETdR, was incorporated into the cell nucleus during synthesis and provided intranuclear beta radiation. Tritium labeled cells were studied with autoradiographic methods; cell cycle kinetics were determined and cell functions modified by radiation dosage or by drugs were also evaluated. The long term program has included; (1) effects of radiation on cell replication and the correlation with incorporated dose levels, (2) radiation induced changes in cell function, viz., the response of beta irradiated spleen lymphocytes to antigenic stimulation by sheep red blood cells (SRBC), (3) kinetics of tumor and normal cell replication; and (4) megakaryocyte formation and modification by radiomimetic drugs.

Infrared spectroscopic studies of myeloid leukemia (ML-1) cells at different phases of the cell cycle

NASA Astrophysics Data System (ADS)

Boydston-White, Susie; Diem, Max

1999-06-01

Advances in infrared spectroscopic methodology permit excellent infrared spectra to be collected from objects as small as single human cells. These advances have lead to an increased interest of the use of infrared spectroscopy as a medical diagnostic tool. Infrared spectra of myeloid leukemia (ML-1) cells are reported for cells derived from an asynchronous, exponentially-growing culture, as well as for cells that were fractionated according to their stage within the cell division cycle. The observed results suggest that the cells' DNA is detectable by infrared spectroscopy mainly when the cell is in the S phase, during the replication of DNA. In the G1 and G2 phases, the DNA is so tightly packed in the nucleus that it appears opaque to infrared radiation. Consequently, the nucleic acid spectral contributions in the G1 and G2 phases would be mostly that of cytoplasmic RNA. These results suggest that infrared spectral changes observed earlier between normal and abnormal cells may have been due to different distributions of cells within the stages of the cell division cycle.

Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs

NASA Technical Reports Server (NTRS)

Schatten, H.; Chakrabarti, A.

1998-01-01

This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their ability to reform normal mitotic figures. These results indicate that chloral hydrate and diazepam affect centrosome structure which results in the inability to reform normal microtubule formations and causes abnormal fertilization and mitosis.

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.

Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

PubMed Central

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

2014-01-01

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

[Recent research advance on bone marrow microenvironment-mediated leukemia drug resistant mechanism].

PubMed

Fu, Bing; Ling, Yan-Juan

2011-06-01

The bone marrow microenvironment consists of bone marrow stromal cells, osteoblasts and osteoclasts which facilities the survival, differentiation and proliferation of hematopoietic cells through secreting soluble factors and extracellular matrix proteins that mediate these functions. This environment not only supports the growth of normal and malignant hematopoietic cells, but also protects them against the damage from chemotherapeutic agents through the secretion of soluble cytokines, cell adhesion, up-regulation of resistant genes and changes of cell cycle. In this review, the research advances on drug-resistance mechanisms mediated by bone marrow microenvironment are summarized briefly, including soluble factors mediating drug resistance, intercellular adhesion inducing drug resistance, up-regulation of some drug resistance genes, regulation in metabolism of leukemic cells, changes in cell cycles of tumor cells and so on.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML

PubMed Central

Bolli, Niccolò; Rhodes, Jennifer; Abdel-Wahab, Omar I.; Levine, Ross; Hedvat, Cyrus V.; Stone, Richard; Khanna-Gupta, Arati; Sun, Hong; Kanki, John P.; Gazda, Hanna T.; Beggs, Alan H.; Cotter, Finbarr E.

2011-01-01

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies. PMID:21653321

Immunocyte responses of mouse exposure by low dose 12C6+ ion beam

NASA Astrophysics Data System (ADS)

Dang, B. R.

High LET radiations such as heavy ion or neutron have an increased biological effectiveness compared to low LET radiations for cell killing cell cycle perturbations and genetic instability In this paper we investigate the peripheral blood lymphocytes thymus cell and spleen lymphocytes cycle effects of exposure with different dose of 73 74MeV u 12 C 6 ion on mouse These BalB C were irradiated with 39cGy 55cGy and 1Gy of 12 C 6 ion at 20cGy min The cell cycle and apoptosis were determined by flow cytometry and the thymus and spleen index were measured by weight When these mice were irradiated by 12 C 6 the cycle of immunocyte had some changes and the percentage of apoptosis increased with doses increasing irradiation especially blood lymphocyte It might be suggested that irradiated by 12 C 6 total-body can result in more damage for immune system than normal irradiation

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line.

PubMed

Chappell, Patrick E; White, Rachel S; Mellon, Pamela L

2003-12-03

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

Selenoprotein P Inhibits Radiation-Induced Late Reactive Oxygen Species Accumulation and Normal Cell Injury

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

Eckers, Jaimee C.; Kalen, Amanda L.; Xiao, Wusheng

2013-11-01

Purpose: Radiation is a common mode of cancer therapy whose outcome is often limited because of normal tissue toxicity. We have shown previously that the accumulation of radiation-induced late reactive oxygen species (ROS) precedes cell death, suggesting that metabolic oxidative stress could regulate cellular radiation response. The purpose of this study was to investigate whether selenoprotein P (SEPP1), a major supplier of selenium to tissues and an antioxidant, regulates late ROS accumulation and toxicity in irradiated normal human fibroblasts (NHFs). Methods and Materials: Flow cytometry analysis of cell viability, cell cycle phase distribution, and dihydroethidium oxidation, along with clonogenic assays,more » were used to measure oxidative stress and toxicity. Human antioxidant mechanisms array and quantitative real-time polymerase chain reaction assays were used to measure gene expression during late ROS accumulation in irradiated NHFs. Sodium selenite addition and SEPP1 overexpression were used to determine the causality of SEPP1 regulating late ROS accumulation and toxicity in irradiated NHFs. Results: Irradiated NHFs showed late ROS accumulation (4.5-fold increase from control; P<.05) that occurs after activation of the cell cycle checkpoint pathways and precedes cell death. The mRNA levels of CuZn- and Mn-superoxide dismutase, catalase, peroxiredoxin 3, and thioredoxin reductase 1 increased approximately 2- to 3-fold, whereas mRNA levels of cold shock domain containing E1 and SEPP1 increased more than 6-fold (P<.05). The addition of sodium selenite before the radiation treatment suppressed toxicity (45%; P<.05). SEPP1 overexpression suppressed radiation-induced late ROS accumulation (35%; P<.05) and protected NHFs from radiation-induced toxicity (58%; P<.05). Conclusion: SEPP1 mitigates radiation-induced late ROS accumulation and normal cell injury.« less

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding

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

Freund, R.; Bauer, P.H.; Benjamin, T.L.

1994-11-01

The authors have examined the growth properties of polyomavirus large T-antigen mutants that ar unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts for mouse embryos that carry a homozygous knockout of the RB gene are permissive, whilemore » those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G{sub 0} or G{sub 1} through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle. 48 refs., 6 figs., 5 tabs.« less

Protein tyrosine phosphatase of liver regeneration-1 is required for normal timing of cell cycle progression during liver regeneration

PubMed Central

Jiao, Yang; Ye, Diana Z.; Li, Zhaoyu; Teta-Bissett, Monica; Peng, Yong; Taub, Rebecca; Greenbaum, Linda E.

2014-01-01

Protein tyrosine phosphatase of liver regeneration-1 (Prl-1) is an immediate-early gene that is significantly induced during liver regeneration. Several in vitro studies have suggested that Prl-1 is important for the regulation of cell cycle progression. To evaluate its function in liver regeneration, we ablated the Prl-1 gene specifically in mouse hepatocytes using the Cre-loxP system. Prl-1 mutant mice (Prl-1loxP/loxP;AlfpCre) appeared normal and fertile. Liver size and metabolic function in Prl-1 mutants were comparable to controls, indicating that Prl-1 is dispensable for liver development, postnatal growth, and hepatocyte differentiation. Mutant mice demonstrated a delay in DNA synthesis after 70% partial hepatectomy, although ultimate liver mass restoration was not affected. At 40 h posthepatectomy, reduced protein levels of the cell cycle regulators cyclin E, cyclin A2, cyclin B1, and cyclin-dependent kinase 1 were observed in Prl-1 mutant liver. Investigation of the major signaling pathways involved in liver regeneration demonstrated that phosphorylation of protein kinase B (AKT) and signal transducer and activator of transcription (STAT) 3 were significantly reduced at 40 h posthepatectomy in Prl-1 mutants. Taken together, this study provides evidence that Prl-1 is required for proper timing of liver regeneration after partial hepatectomy. Prl-1 promotes G1/S progression via modulating expression of several cell cycle regulators through activation of the AKT and STAT3 signaling pathway. PMID:25377314

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

PubMed Central

Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

2015-01-01

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K i<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug. PMID:26398286

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility.

PubMed

Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B; Murray, Brion W

2015-01-01

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2-6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.

The "Yin" and "Yang" of Cell Cycle Progression and Differentiation in the Oligodendroglial Lineage

ERIC Educational Resources Information Center

Nguyen, Laurent; Borgs, Laurence; Vandenbosch, Renaud; Mangin, Jean-Marie; Beukelaers, Pierre; Moonen, Gustave; Gallo, Vittorio; Malgrange, Brigitte; Belachew, Shibeshih

2006-01-01

In white matter disorders such as leukodystrophies (LD), periventricular leucomalacia (PVL), or multiple sclerosis (MS), the hypomyelination or the remyelination failure by oligodendrocyte progenitor cells involves errors in the sequence of events that normally occur during development when progenitors proliferate, migrate through the white…

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

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

2015-04-01

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

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

PubMed

Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

2015-09-08

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle.

PubMed

Ramasamy, Rajesh; Fazekasova, Henrietta; Lam, Eric W-F; Soeiro, Inês; Lombardi, Giovanna; Dazzi, Francesco

2007-01-15

Mesenchymal stem cells (MSCs) play a crucial role in hematopoietic development and have been shown to exert a powerful immunosuppressive effect. In this study, we investigated the effect of bone marrow MSC on the differentiation and function of peripheral blood monocytes into dendritic cells (DCs). Human MSCs, generated from normal bone marrow, were added to peripheral blood monocytes stimulated in vitro with granulocyte-macrophage colony stimulating factor and interleukin-4 to become DCs. Monocytes were then examined for the expression of markers characteristic of DCs and their ability to stimulate allogeneic T cells. In addition, the effect of MSCs on the cell cycle of monocyte-derived DCs and the expression of various cell cycle proteins were analyzed by cytometric analysis and Western blotting with specific antibodies. MSCs blocked the differentiation of monocytes into DCs and impaired their antigen-presenting ability. This resulted from a block of monocytes from entering the G1 phase of the cell cycle with a progressive number of cells accumulating in the G0 phase. Cyclin D2 was downregulated. However, differently from what was observed in T-cells stimulated in the presence of MSCs, the expression of p27 was found decreased, suggesting the involvement of similar but not identical pathways. We conclude that MSCs impair monocyte differentiation and function by interfering with the cell cycle. These findings imply that MSC-induced immunosuppression might be a side product of a more general antiproliferative effect.

RGC-32 is a novel regulator of the T-lymphocyte cell cycle.

PubMed

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

2015-06-01

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

Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

NASA Astrophysics Data System (ADS)

Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

2012-07-01

Spaceflight factors, including microgravity and space radiation, have many detrimental short-term effects on human physiology, including muscle and bone degradation, and immune system dysfunction. The long-term progression of these physiological effects is still poorly understood, and a serious concern for long duration spaceflight missions. We hypothesized that some of the degenerative effects of spaceflight may be caused in part by an inability of stem cells to proliferate and differentiate normally resulting in an impairment of tissue regenerative processes. Furthermore, we hypothesized that long-term bone tissue degeneration in space may be mediated by activation of the p53 signaling network resulting in cell cycle arrest and/or apoptosis in osteoprogenitors. In our analyses we found that spaceflight caused significant bone loss in the weight-bearing bones of mice with a 6.3% reduction in bone volume and 11.9% decrease in bone thickness associated with increased osteoclastic activity. Along with this rapid bone loss we also observed alterations in the cell cycle characterized by an increase in the Cdkn1a/p21 cell cycle arrest molecule independent of Trp53. Overexpression of Cdkn1a/p21 was localized to osteoblasts lining the periosteal surface of the femur and chondrocytes in the head of the femur, suggesting an inhibition of proliferation in two key regenerative cell types of the femur in response to spaceflight. Additionally we found overexpression of several matrix degradation molecules including MMP-1a, 3 and 10, of which MMP-10 was localized to osteocytes within the shaft of the femur. This, in conjunction with 40 nm resolution synchrotron nano-Computed Tomography (nano-CT) observations of an increase in osteocyte lacunae cross-sectional area, perimeter and a decrease in circularity indicates a potential role for osteocytic osteolysis in the observed bone degeneration in spaceflight. To further investigate the genetic response of bone to mechanical unloading in spaceflight, we conducted genome wide microarray analysis of total RNA isolated from the mouse pelvis. Specifically, 16 week old mice were subjected to 15 days spaceflight onboard NASA's STS-131 space shuttle mission. The pelvis of the mice was dissected, the bone marrow was flushed and the bones were briefly stored in RNAlater. The pelvii were then homogenized, and RNA was isolated using TRIzol. RNA concentration and quality was measured using a Nanodrop spectrometer, and 0.8% agarose gel electrophoresis. Samples of cDNA were analyzed using an Affymetrix GeneChip\\S Gene 1.0 ST (Sense Target) Array System for Mouse and GenePattern Software. We normalized the ST gene arrays using Robust Multichip Average (RMA) normalization, which summarizes perfectly matched spots on the array through the median polish algorithm, rather than normalizing according to mismatched spots. We also used Limma for statistical analysis, using the BioConductor Limma Library by Gordon Smyth, and differential expression analysis to identify genes with significant changes in expression between the two experimental conditions. Finally we used GSEApreRanked for Gene Set Enrichment Analysis (GSEA), with Kolmogorov-Smirnov style statistics to identify groups of genes that are regulated together using the t-statistics derived from Limma. Preliminary results show that 6,603 genes expressed in pelvic bone had statistically significant alterations in spaceflight compared to ground controls. These prominently included cell cycle arrest molecules p21, and p18, cell survival molecule Crbp1, and cell cycle molecules cyclin D1, and Cdk1. Additionally, GSEA results indicated alterations in molecular targets of cyclin D1 and Cdk4, senescence pathways resulting from abnormal laminin maturation, cell-cell contacts via E-cadherin, and several pathways relating to protein translation and metabolism. In total 111 gene sets out of 2,488, about 4%, showed statistically significant set alterations. These alterations indicate significant impairment of normal cellular function in the mechanically unloaded environment of space and could provide important genetic insight into the observed uncoupling of bone formation and resorption in space.

Effects of air transient spark discharge and helium plasma jet on water, bacteria, cells, and biomolecules.

PubMed

Hensel, Karol; Kučerová, Katarína; Tarabová, Barbora; Janda, Mário; Machala, Zdenko; Sano, Kaori; Mihai, Cosmin Teodor; Ciorpac, Mitică; Gorgan, Lucian Dragos; Jijie, Roxana; Pohoata, Valentin; Topala, Ionut

2015-06-06

Atmospheric pressure DC-driven self-pulsing transient spark (TS) discharge operated in air and pulse-driven dielectric barrier discharge plasma jet (PJ) operated in helium in contact with water solutions were used for inducing chemical effects in water solutions, and the treatment of bacteria (Escherichia coli), mammalian cells (Vero line normal cells, HeLa line cancerous cells), deoxyribonucleic acid (dsDNA), and protein (bovine serum albumin). Two different methods of water solution supply were used in the TS: water electrode system and water spray system. The effects of both TS systems and the PJ were compared, as well as a direct exposure of the solution to the discharge with an indirect exposure to the discharge activated gas flow. The chemical analysis of water solutions was performed by using colorimetric methods of UV-VIS absorption spectrophotometry. The bactericidal effects of the discharges on bacteria were evaluated by standard microbiological plate count method. Viability, apoptosis and cell cycle were assessed in normal and cancerous cells. Viability of cells was evaluated by trypan blue exclusion test, apoptosis by Annexin V-FITC/propidium iodide assay, and cell cycle progression by propidium iodide/RNase test. The effect of the discharges on deoxyribonucleic acid and protein were evaluated by fluorescence and UV absorption spectroscopy. The results of bacterial and mammalian cell viability, apoptosis, and cell cycle clearly show that cold plasma can inactivate bacteria and selectively target cancerous cells, which is very important for possible future development of new plasma therapeutic strategies in biomedicine. The authors found that all investigated bio-effects were stronger with the air TS discharge than with the He PJ, even in indirect exposure.

The Biological Role of Nestin(+)-Cells in Physiological and Pathological Cardiovascular Remodeling

PubMed Central

Calderone, Angelino

2018-01-01

The intermediate filament protein nestin was identified in diverse populations of cells implicated in cardiovascular remodeling. Cardiac resident neural progenitor/stem cells constitutively express nestin and following an ischemic insult migrate to the infarct region and participate in angiogenesis and neurogenesis. A modest number of normal adult ventricular fibroblasts express nestin and the intermediate filament protein is upregulated during the progression of reparative and reactive fibrosis. Nestin depletion attenuates cell cycle re-entry suggesting that increased expression of the intermediate filament protein in ventricular fibroblasts may represent an activated phenotype accelerating the biological impact during fibrosis. Nestin immunoreactivity is absent in normal adult rodent ventricular cardiomyocytes. Following ischemic damage, the intermediate filament protein is induced in a modest population of pre-existing adult ventricular cardiomyocytes bordering the peri-infarct/infarct region and nestin(+)-ventricular cardiomyocytes were identified in the infarcted human heart. The appearance of nestin(+)-ventricular cardiomyocytes post-myocardial infarction (MI) recapitulates an embryonic phenotype and depletion of the intermediate filament protein inhibits cell cycle re-entry. Recruitment of the serine/threonine kinase p38 MAPK secondary to an overt inflammatory response after an ischemic insult may represent a seminal event limiting the appearance of nestin(+)-ventricular cardiomyocytes and concomitantly suppressing cell cycle re-entry. Endothelial and vascular smooth muscle cells (VSMCs) express nestin and upregulation of the intermediate filament protein may directly contribute to vascular remodeling. This review will highlight the biological role of nestin(+)-cells during physiological and pathological remodeling of the heart and vasculature and discuss the phenotypic advantage attributed to the intermediate filament protein. PMID:29492403

Megakaryocyte polyploidization is associated with decreased expression of polo-like kinase (PLK).

PubMed

Yagi, M; Roth, G J

2006-09-01

During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mouse polo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.

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.

Biomedical Applications of the Cold Atmospheric Plasma: Cell Responses

NASA Astrophysics Data System (ADS)

Volotskova, Olga

Current breakthrough research on cold atmospheric plasma (CAP) demonstrates that CAP has great potential in various areas, including medicine and biology, thus providing a new tool for living tissue treatment. Depending on the configuration the cold plasma sources can be used in the following areas: wound healing, skin diseases, hospital hygiene, sterilization, antifungal treatments, dental care, cosmetics targeted cell/tissue removal, and cancer treatments. This dissertation is focused on the studies of biomedical applications of cold atmospheric plasma jet based on helium flow and resultant cell responses to the cold plasma treatment. The studies were carried out on extra-cellular and intra-cellular levels in vitro. The main practical applications are wound healing and alternative to existing cancer therapy methods, areas of great interest and significant challenges. The CAP jet was built in the Micropropulsion and Nanotechnology Laboratory of Dr. Michael Keidar, as a part of multidisciplinary collaboration with the GW Medical School (Dr. M.A. Stepp) concerned with plasma medicine and bioengineering studies. Normal and cancer cells have two fundamental behavioral properties, proliferation and motility, which can be evaluated through cell migration rates and cell cycle progression. Various microscopic, spectroscopic and flow cytometry techniques were used to characterize cell responses to the cold plasma treatment. It was found that CAP effect on the cells is localized within the area of the treatment (of around ˜ 5mm in diameter). The migration rates of the normal skin cells can be reduced up to ˜ 40%. However, depending on the cell type the required treatment time is different, thus differential treatment of various cells presented in tissue is possible. The CAP effect on the migration was explained through the changes of the cell surface proteins/integrins. It was also found that normal and cancer cells respond differently to the CAP treatment under the same experimental conditions. CAP is currently being evaluated as a new highly selective alternative addition to existing cancer therapies. It was shown that the increased sensitivity of cancer cells to CAP treatment is caused by differences in the distribution of cancer cells and normal cells within the cell cycle. It was also shown that the expression of γH2A.X (pSer139), an oxidative stress reporter indicating S-phase damage, is enhanced specifically within CAP treated cells in the S phase of the cell cycle together with significant decrease in EdU-signal of DNA-replicating cells. Thus, newly developed CAP technology was proven to be of a great interest for practical applications in the areas of wound healing and cancer treatment. The identification and explanation of the mechanisms by which CAP affects the cells was presented.

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

PubMed Central

Burkhart, Deborah L.; Wirt, Stacey E.; Zmoos, Anne-Flore; Kareta, Michael S.; Sage, Julien

2010-01-01

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells. PMID:20585628

TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation.

PubMed

Bravo, Susana B; Pampín, Sandra; Cameselle-Teijeiro, José; Carneiro, Carmen; Domínguez, Fernando; Barreiro, Francisco; Alvarez, Clara V

2003-10-30

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.

Requirement of ClC-3 in G0/G1 to S Phase Transition Induced by IGF-1 via ERK1/2-Cyclins Cascade in Multiple Myeloma Cells.

PubMed

Du, Yu; Tu, Yong-Sheng; Tang, Yong-Bo; Huang, Yun-Ying; Zhou, Fang-Min; Tian, Tian; Li, Xiao-Yan

2018-06-01

ClC-3 is involved in the proliferation and migration of several cancer cells. However, ClC-3 expression and its role of cell-cycle control in multiple myeloma (MM) has not yet been investigated. MM cells were treated with different concentrations of IGF (30, 100, 300 ng/mL), and their proliferation was examined by CCK-8. The effects of ClC-3 on cell cycle progression was detected by flow cytometry. Western blot was used to analyze the relative levels of ClC3, CD138, P21, P27, CDK, p-Erk1/2, and t-Erk1/2 protein expression. Transfection of RPMI8226 with gpClC-3 cDNA and siRNA alters the expression of ClC-3. We compared the expression of ClC-3 in primary myeloma cells and in MM cell lines (U266 and RPMI8266) with that in normal plasma cells (PCs) from normal subjects and found that myeloma cells from patients and MM cell lines had significantly higher expression of ClC-3. Additionally, silencing of ClC-3 with the small interfering RNA (siRNA) that targets human ClC-3 decreased proliferation of RPMI8226 after IGF-1 treatment and slowed cell cycle progression from G0/G1 to S phase, which was associated with diminished phosphorylation of ERK1/2, down-expression of cyclin E, cyclin D1 and up-regulation of p27 and p21. By contrast, overexpression of ClC-3 potentiated cell proliferation induced by IGF-1, raised the percentage of S phase cells, enhanced phosphorylation of ERK1/2, downregulated p27 and p21 and upregulated cyclin E and cyclin D1. ClC-3 accelerated G0/G1 to S phase transition in the cell cycle by modulating ERK1/2 kinase activity and expression of G1/S transition related proteins, making ClC-3 an attractive therapeutic target in MM.

Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

PubMed Central

Hunziker, E B; Wagner, J; Zapf, J

1994-01-01

Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells. Images PMID:8132746

Effects of Different Zinc Species on Cellar Zinc Distribution, Cell Cycle, Apoptosis and Viability in MDAMB231 Cells.

PubMed

Wang, Yan-hong; Zhao, Wen-jie; Zheng, Wei-juan; Mao, Li; Lian, Hong-zhen; Hu, Xin; Hua, Zi-chun

2016-03-01

Intracellular metal elements exist in mammalian cells with the concentration range from picomoles per litre to micromoles per litre and play a considerable role in various biological procedures. Element provided by different species can influence the availability and distribution of the element in a cell and could lead to different biological effects on the cell's growth and function. Zinc as an abundant and widely distributed essential trace element, is involved in numerous and relevant physiological functions. Zinc homeostasis in cells, which is regulated by metallothioneins, zinc transporter/SLC30A, Zrt-/Irt-like proteins/SLC39A and metal-response element-binding transcription factor-1 (MTF-1), is crucial for normal cellular functioning. In this study, we investigated the influences of different zinc species, zinc sulphate, zinc gluconate and bacitracin zinc, which represented inorganic, organic and biological zinc species, respectively, on cell cycle, viability and apoptosis in MDAMB231 cells. It was found that the responses of cell cycle, apoptosis and death to different zinc species in MDAMB231 cells are different. Western blot analysis of the expression of several key proteins in regulating zinc-related transcription, cell cycle, apoptosis, including MTF-1, cyclin B1, cyclin D1, caspase-8 and caspase-9 in treated cells further confirmed the observed results on cell level.

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.

Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells.

PubMed

Rajavel, Tamilselvam; Mohankumar, Ramar; Archunan, Govindaraju; Ruckmani, Kandasamy; Devi, Kasi Pandima

2017-06-13

Lung cancer is the leading cause of cancer related deaths both in developed and developing countries. Since majority of the existing therapeutic methods harms both normal and malignant cells, a viable alternative is to switch into safe and beneficial traditional medicinal plants. Hence the present study was framed to identify selective anti-lung cancer agents from the medicinal plant Grewia tiliaefolia (GT). Cell viability experiments showed that benzene extract of GT (BGT) leaf effectively inhibited the growth of A549 cells, while being non-toxic to normal human lung L132 and PBMC cells. Ames and comet assays demonstrated that BGT is of non-mutagenic and non-genotoxic nature in untransformed cells. The hematological and histopathological profiles of the in vivo acute and sub-acute toxicity studies demonstrated that BGT is safe and tolerable. Importantly, western blot analysis and Annexin V-FITC staining confirmed that BGT promotes mitochondrial dependent apoptotic cell death in A549 cells by arresting cell cycle at G2/M phase. Bio-assay guided fractionation revealed the presence of phytosteols (β-sitosterol and daucosterol) which significantly inhibited the growth of A549 cells both alone and in combination. This study warrants that these phytosterols in alone or in combination can be considered as safe and potential drug candidates for lung cancer treatment.

Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma

PubMed Central

Yang, Qingshan; Chen, Lisa S.; Neelapu, Sattva S.; Miranda, Roberto N.; Medeiros, L. Jeffrey

2012-01-01

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small mol-ecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL. PMID:22955922

Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma.

PubMed

Yang, Qingshan; Chen, Lisa S; Neelapu, Sattva S; Miranda, Roberto N; Medeiros, L Jeffrey; Gandhi, Varsha

2012-10-25

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small molecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL.

Teaching the basics of cancer metabolism: Developing antitumor strategies by exploiting the differences between normal and cancer cell metabolism.

PubMed

Kalyanaraman, Balaraman

2017-08-01

This review of the basics of cancer metabolism focuses on exploiting the metabolic differences between normal and cancer cells. The first part of the review covers the different metabolic pathways utilized in normal cells to generate cellular energy, or ATP, and the glycolytic intermediates required to build the cellular machinery. The second part of the review discusses aerobic glycolysis, or the Warburg effect, and the metabolic reprogramming involving glycolysis, tricarboxylic acid cycle, and glutaminolysis in the context of developing targeted inhibitors in cancer cells. Finally, the selective targeting of cancer mitochondrial metabolism using positively charged lipophilic compounds as potential therapeutics and their ability to mitigate the toxic side effects of conventional chemotherapeutics in normal cells are discussed. I hope this graphical review will be useful in helping undergraduate, graduate, and medical students understand how investigating the basics of cancer cell metabolism could provide new insight in developing potentially new anticancer treatment strategies. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

L'effet de p53 sur la radiosensibilité des cellules humaines normales et cancéreuses

NASA Astrophysics Data System (ADS)

Little, J. B.; Li, C. Y.; Nagasawa, H.; Huang, H.

1998-04-01

The radiosensitivity of normal human fibroblasts in p53 dependent and associated with the loss of cells from the cycling population as the result of an irreversible G1 arrest; cells lacking normal p53 function show no arrest and are more radioresistant. Under conditions in which the repair potentially lethal radiation damage is facilitated, the fraction of cells arrested in G1 is reduced and survival is enhanced. The response of human tumor cells differs significantly. The radiation-induced G1 arrest is minimal or absent in p53+ tumor cells, and loss of normal p53 function has no consistent effect on their radiosensitivity. These results suggest that p53 status may not be a useful predictive marker for the response of human solid tumors to radiation therapy. La radiosensibilité des fibroblastes diploïdes humains est liée à l'expression de p53, et à la perte de cellules en cycle résultant d'un arrêt irréversible en phase G1 ; dans les cellules n'ayant pas une fonction p53 normale, on ne constate aucun arrêt, et elles sont plus radio-résistantes. Dans des conditions favorables à la réparation de lésions potentiellement léthales dues à l'irradiation, la proportion de cellules bloquées en phase G1 baisse, et les chances de survie sont accrues. Bien différente est la réaction des cellules cancéreuses humaines. Le blocage par irradiation en phase G1 est minime ou inexistant dans les cellules cancéreuses p53^+, et la perte de la fonction normale p53 n'a pas d'effet constant sur leur radiosensibilité. Ces résultats laissent penser que l'expression de p53 n'est pas un indice fiable permettant de prévoir la réaction des tumeurs solides à la radiothérapie.

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

NASA Astrophysics Data System (ADS)

Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

2012-09-01

The long-term ``fate'' of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability.

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

PubMed Central

Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

2012-01-01

The long-term “fate” of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability. PMID:22966418

A long noncoding RNA UCA1 promotes proliferation and predicts poor prognosis in glioma.

PubMed

Zhao, W; Sun, C; Cui, Z

2017-06-01

Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma.

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

PubMed

Lerit, Dorothy A; Jordan, Holly A; Poulton, John S; Fagerstrom, Carey J; Galletta, Brian J; Peifer, Mark; Rusan, Nasser M

2015-07-06

Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Redox cycling of Cu(II) by 6-mercaptopurine leads to ROS generation and DNA breakage: possible mechanism of anticancer activity.

PubMed

Rehman, Sayeed Ur; Zubair, Haseeb; Sarwar, Tarique; Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Nehar, Shamshun; Tabish, Mohammad

2015-02-01

6-Mercaptopurine (6MP) is a well-known purine antimetabolite used to treat childhood acute lymphoblastic leukemia and other diseases. Cancer cells as compared to normal cells are under increased oxidative stress and show high copper level. These differences between cancer cells and normal cells can be targeted to develop effective cancer therapy. Pro-oxidant property of 6MP in the presence of metal ions is not well documented. Redox cycling of Cu(II) to Cu(I) was found to be efficiently mediated by 6MP. We have performed a series of in vitro experiments to demonstrate the pro-oxidant property of 6MP in the presence of Cu(II). Studies on human lymphocytes confirmed the DNA damaging ability of 6MP in the presence of Cu(II). Since 6MP possesses DNA damaging ability by producing reactive oxygen species (ROS) in the presence of Cu(II), it may also possess apoptosis-inducing activity by involving endogenous copper ions. Essentially, this would be an alternative and copper-dependent pathway for anticancer activity of 6MP.

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii.

PubMed

Dhara, Animesh; de Paula Baptista, Rodrigo; Kissinger, Jessica C; Snow, E Charles; Sinai, Anthony P

2017-11-21

The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated o varian tu mor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture. IMPORTANCE Replication by Toxoplasma gondii can occur by 3 distinct cell cycle architectures. Endodyogeny is used by asexual stages, while a hybrid of schizogony and endopolygeny is used by merozoites in the definitive feline host. Here, we establish that the disruption of an o varian- tu mor (OTU) family deubiquitinase, TgOTUD3A, in tachyzoites results in dysregulation of the mechanism controlling the selection of replication strategy in a subset of parasites. The mechanistic basis for these altered cell cycles lies in the unique biology of the bipartite centrosome that is associated with the transient loss of stoichiometry between the inner and outer centrosome cores in the TgOTUD3A-KO mutant. This highlights the importance of ubiquitin-mediated regulation in the transition from the nuclear to the budding phases of the cell cycle and provides new mechanistic insights into the regulation of the organization of the apicomplexan cell cycle. Copyright © 2017 Dhara et al.

Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts.

PubMed

Markopoulos, Georgios S; Roupakia, Eugenia; Tokamani, Maria; Vartholomatos, George; Tzavaras, Theodore; Hatziapostolou, Maria; Fackelmayer, Frank O; Sandaltzopoulos, Raphael; Polytarchou, Christos; Kolettas, Evangelos

2017-10-01

Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G 1 /S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G 1 /S and G 2 /M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

The cytotoxic, apoptotic and oxidative effects of carbonic anhydrase IX inhibitor on colorectal cancer cells.

PubMed

Tülüce, Yasin; Ahmed, Bewar Ali; Koyuncu, İsmail; Durgun, Mustafa

2018-04-01

Colorectal cancer (CRC) is the third most common tumor, malignant and has developed one of the main reasons of cancer mortality. According to studies conducted recently; carbonic anhydrase 9 (CAIX) is an especially attractive target for cancer therapy, in part since it is limited way expressed in normal tissues on the other hand in a wide variety of solid neoplasia are overexpressed. The aim of this study was to appreciate the effects of CAIX inhibitor, namely novel synthesized sulfonamide derivative (H-4i) with high affinity for CAIX, in CAIX-positive human colorectal cancer cell (HT-29) and CAIX-negative human normal embryonic kidney cell line (HEK-293). For this reason, we planned to investigate apoptotic, cytotoxic and oxidative stress activity of H-4i on HT-29 and HEK-293 cell lines. Cell viability determined by WST-1 assay afterwards IC 50 values, apoptosis and cell cycle induction measured by flow cytometric analysis, intracellular free radical induction performed by reactive oxygen species (ROS) analyses. The IC 50 value of the sulfonamide derivative compound was found to be very low, especially in HT-29 cells, when compared to human normal cells. This research found that H-4i significantly increased cytotoxicity and ROS production, caused significant signs of apoptosis level. High level of ROS and apoptosis lead to arrest the cell cycle and reduce cell survival. The most obvious finding to emerge from the analysis that novel synthesized sulfonamide derivative H-4i is effective on HT-29 more than HEK-293. Therefore, novel derivative H-4i might be used as an anti-cancer potential compound on CRC.

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

PubMed Central

Johnson, M Cecilia; Torres, Marisa; Alves, Alessandra; Bacallao, Ketty; Fuentes, Ariel; Vega, Margarita; Boric, M Angélica

2005-01-01

Background Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. Methods Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. Results Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05). Conclusion An altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium. PMID:16150151

Cell cycle perturbations and genotoxic effects in human primary fibroblasts induced by low-energy protons and X/gamma-rays.

PubMed

Antoccia, Antonio; Sgura, Antonella; Berardinelli, Francesco; Cavinato, Maria; Cherubini, Roberto; Gerardi, Silvia; Tanzarella, Caterina

2009-09-01

The effect of graded doses of high-linear energy transfer (LET) low-energy protons to induce cycle perturbations and genotoxic damage was investigated in normal human fibroblasts. Furthermore, such effects were compared with those produced by low-LET radiations. HFFF2, human primary fibroblasts were exposed to either protons (LET = 28.5 keV/microm) or X/gamma-rays, and endpoints related to cell cycle kinetics and DNA damage analysed. Following both type of irradiations, unsynchronized cells suffered an inhibition to entry into S-phase for doses of 1-4 Gy and remained arrested in the G(1)-phase for several days. The levels of induction of regulator proteins, such as TP53 and CDKN1A showed a clear LET-dependence. DSB induction and repair as measured by scoring for gamma-H2AX foci indicated that protons, with respect to X-rays, yielded a lower number of DSBs per Gy, which showed a slower kinetics of disappearance. Such result was in agreement with the extent of MN induction in binucleated cells after X-irradiation. No significant differences between the two types of radiations were observed with the clonogenic assay, resulting anyway the slope of gamma-ray curve higher than that the proton one. In conclusion, in normal human primary fibroblasts cell cycle arrest at the G(1)/S transition can be triggered shortly after irradiation and maintained for several hours post-irradiation of both protons and X-rays. DNA damage produced by protons appears less amenable to be repaired and could be transformed in cytogenetic damage in the form of MN.

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies. PMID:22723265

Duration of division-related events in cleaving sand dollar eggs.

PubMed

Rappaport, R; Rappaport, B N

1993-07-01

A minimal mechanism for cytokinesis comprises a stimulus-to-surface contraction, a receptive surface, and a localized surface contractile mechanism. Duration of each is brief and times when they function are predictable. The processes that begin and end the functional period of each component were investigated. Sand dollar blastomeres from the completion of first cleavage to the beginning of fourth cleavage were used. By changing a cell's shape, it was possible to determine whether its capacity to accomplish an activity is restricted to its usual time frame. The first appearance of the furrow was advanced about 5 min by confining the mitotic apparatus in a narrow cytoplasmic cylinder. The period when the mitotic apparatus induces furrowing was prolonged about 18 min by moving the mitotic apparatus in an elongate cell each time the furrow appeared. The period of active furrowing was prolonged to about 21.8 min by pushing the mitotic apparatus close to the cell margin and then stretching the region through which the unilateral furrow must pass. In relation to normal division cycle events, results showed that each event of cytokinesis can operate both before and after its normal active period. Components of the mechanism are capable of functioning for about half the period of the division cycle. Normal timing of events may be determined by geometrical factors and the normal consequences of each activity.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

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.

Cytotoxic effects of ultra-diluted remedies on breast cancer cells.

PubMed

Frenkel, Moshe; Mishra, Bal Mukund; Sen, Subrata; Yang, Peiying; Pawlus, Alison; Vence, Luis; Leblanc, Aimee; Cohen, Lorenzo; Banerji, Pratip; Banerji, Prasanta

2010-02-01

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Cellular Effect of Curcumin and Citral Combination on Breast Cancer Cells: Induction of Apoptosis and Cell Cycle Arrest.

PubMed

Patel, Pinaki B; Thakkar, Vasudev R; Patel, Jagdish S

2015-09-01

The unmanageable side effects caused by current chemotherapy regimens to treat cancer are an unresolved problem. Although many phytonutrients are useful as chemoprevention without side effects, their effects are slower and smaller than conventional chemotherapy. In the present work, we examined the cumulative effect of two phytonutrients, curcumin and citral, on breast cancer cell lines and compared their effect with the known chemotherapy regimen of cyclophosphamide, methotrexate, and 5-fluorouracil. Using cultured breast cancer and normal epithelial cells, the cytotoxic and apoptotic effect of curcumin and citral was evaluated in vitro. The synergistic effect of curcumin and citral was calculated by a combination index study using the method by Chou and Talalay. Cell death pathways and mechanisms were analyzed by measuring intracellular reactive oxygen species (ROS) and apoptotic protein levels. Curcumin and citral caused dose and time dependent cell death and showed a synergistic effect at effective concentration EC50 and above concentrations in breast cancer cells without disturbing normal breast epithelial cells. With combination curcumin and citral treatment, apoptosis induction and cell cycle arrest at G0/G1 phase in breast cancer cells were observed. Curcumin and citral generated ROS and activated p53 and poly (ADP-ribose) polymerase-1 mediated apoptotic pathways. The results of this study suggest that curcumin and citral in combination may be a useful therapeutic intervention for breast cancer.

A high-resolution transcriptome map of cell cycle reveals novel connections between periodic genes and cancer

PubMed Central

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

2016-01-01

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

Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle.

PubMed

Proseus, Timothy E; Boyer, John S

2012-06-01

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

A yeast gene essential for regulation of spindle pole duplication.

PubMed Central

Baum, P; Yip, C; Goetsch, L; Byers, B

1988-01-01

In eucaryotic cells, duplication of spindle poles must be coordinated with other cell cycle functions. We report here the identification in Saccharomyces cerevisiae of a temperature-sensitive lethal mutation, esp1, that deregulates spindle pole duplication. Mutant cells transferred to the nonpermissive temperature became unable to continue DNA synthesis and cell division but displayed repeated duplication of their spindle pole bodies. Although entry into this state after transient challenge by the nonpermissive temperature was largely lethal, rare survivors were recovered and found to have become increased in ploidy. If the mutant cells were held in G0 or G1 during exposure to the elevated temperature, they remained viable and maintained normal numbers of spindle poles. These results suggest dual regulation of spindle pole duplication, including a mechanism that promotes duplication as cells enter the division cycle and a negative regulatory mechanism, controlled by ESP1, that limits duplication to a single occurrence in each cell division cycle. Tetrad analysis has revealed that ESP1 resides at a previously undescribed locus on the right arm of chromosome VII. Images PMID:3072479

Comparing shut-down strategies for proton exchange membrane fuel cells

NASA Astrophysics Data System (ADS)

Oyarce, Alejandro; Zakrisson, Erik; Ivity, Matthew; Lagergren, Carina; Ofstad, Axel Baumann; Bodén, Andreas; Lindbergh, Göran

2014-05-01

Application of system strategies for mitigating carbon corrosion of the catalyst support in proton exchange fuel cells (PEMFCs) is a requirement for PEMFC systems, especially in the case of systems for transport application undergoing thousands of start-ups and shut-downs (SU/SD) during its lifetime. This study compares several of the most common shut-down strategies for 1100 cycles SU/SD cycles at 70 °C and 80% RH using commercially available fuel cell components. Each cycle simulates a prolonged shut-down, i.e. finishing each cycle with air filled anode and cathode. Furthermore, all start-ups are unprotected, i.e. introducing the H2 rich gas into an air filled anode. Finally, each cycle also includes normal fuel cell operation at 0.5 A cm-2 using synthetic reformate/air. H2 purge of the cathode and O2 consumption using a load were found to be the most effective strategies. The degradation rate using the H2 purge strategy was 23 μV cycle-1 at 0.86 A cm-2 using H2 and air at the anode and cathode, respectively. This degradation rate may be regarded as a generally low value, especially considering that this value also includes the degradation rate caused by unprotected start-ups.

Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells.

PubMed

Schwartz, R S; Musto, S; Fabry, M E; Nagel, R L

1998-12-15

In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer.

PubMed

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

2018-05-30

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

Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death via the Mitochondrial Pathway in AML Cells

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Woessner, Richard; Gross, Stefan; Schober, Wendy D.; Estrov, Zeev; Kantarjian, Hagop; Andreeff, Michael

2013-01-01

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines which express high levels of KSP. Knockdown of p53, overexpression of XIAP, and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP, and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has potential to eradicate AML progenitor cells. PMID:19458629

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 MYC Road to Hearing Restoration

PubMed Central

Kopecky, Benjamin; Fritzsch, Bernd

2012-01-01

Current treatments for hearing loss, the most common neurosensory disorder, do not restore perfect hearing. Regeneration of lost organ of Corti hair cells through forced cell cycle re-entry of supporting cells or through manipulation of stem cells, both avenues towards a permanent cure, require a more complete understanding of normal inner ear development, specifically the balance of proliferation and differentiation required to form and to maintain hair cells. Direct successful alterations to the cell cycle result in cell death whereas regulation of upstream genes is insufficient to permanently alter cell cycle dynamics. The Myc gene family is uniquely situated to synergize upstream pathways into downstream cell cycle control. There are three Mycs that are embedded within the Myc/Max/Mad network to regulate proliferation. The function of the two ear expressed Mycs, N-Myc and L-Myc were unknown less than two years ago and their therapeutic potentials remain speculative. In this review, we discuss the roles the Mycs play in the body and what led us to choose them to be our candidate gene for inner ear therapies. We will summarize the recently published work describing the early and late effects of N-Myc and L-Myc on hair cell formation and maintenance. Lastly, we detail the translational significance of our findings and what future work must be performed to make the ultimate hearing aid: the regeneration of the organ of Corti. PMID:24710525

Estimating division and death rates from CFSE data

NASA Astrophysics Data System (ADS)

de Boer, Rob J.; Perelson, Alan S.

2005-12-01

The division tracking dye, carboxyfluorescin diacetate succinimidyl ester (CFSE) is currently the most informative labeling technique for characterizing the division history of cells in the immune system. Gett and Hodgkin (Nat. Immunol. 1 (2000) 239-244) have proposed to normalize CFSE data by the 2-fold expansion that is associated with each division, and have argued that the mean of the normalized data increases linearly with time, t, with a slope reflecting the division rate p. We develop a number of mathematical models for the clonal expansion of quiescent cells after stimulation and show, within the context of these models, under which conditions this approach is valid. We compare three means of the distribution of cells over the CFSE profile at time t: the mean, [mu](t), the mean of the normalized distribution, [mu]2(t), and the mean of the normalized distribution excluding nondivided cells, .In the simplest models, which deal with homogeneous populations of cells with constant division and death rates, the normalized frequency distribution of the cells over the respective division numbers is a Poisson distribution with mean [mu]2(t)=pt, where p is the division rate. The fact that in the data these distributions seem Gaussian is therefore insufficient to establish that the times at which cells are recruited into the first division have a Gaussian variation because the Poisson distribution approaches the Gaussian distribution for large pt. Excluding nondivided cells complicates the data analysis because , and only approaches a slope p after an initial transient.In models where the first division of the quiescent cells takes longer than later divisions, all three means have an initial transient before they approach an asymptotic regime, which is the expected [mu](t)=2pt and . Such a transient markedly complicates the data analysis. After the same initial transients, the normalized cell numbers tend to decrease at a rate e-dt, where d is the death rate.Nonlinear parameter fitting of CFSE data obtained from Gett and Hodgkin to ordinary differential equation (ODE) models with first-order terms for cell proliferation and death gave poor fits to the data. The Smith-Martin model with an explicit time delay for the deterministic phase of the cell cycle performed much better. Nevertheless, the insights gained from analysis of the ODEs proved useful as we showed by generating virtual CFSE data with a simulation model, where cell cycle times were drawn from various distributions, and then computing the various mean division numbers.

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

PubMed

Stein, G H

1985-10-01

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

Mathematical Model of Naive T Cell Division and Survival IL-7 Thresholds.

PubMed

Reynolds, Joseph; Coles, Mark; Lythe, Grant; Molina-París, Carmen

2013-01-01

We develop a mathematical model of the peripheral naive T cell population to study the change in human naive T cell numbers from birth to adulthood, incorporating thymic output and the availability of interleukin-7 (IL-7). The model is formulated as three ordinary differential equations: two describe T cell numbers, in a resting state and progressing through the cell cycle. The third is introduced to describe changes in IL-7 availability. Thymic output is a decreasing function of time, representative of the thymic atrophy observed in aging humans. Each T cell is assumed to possess two interleukin-7 receptor (IL-7R) signaling thresholds: a survival threshold and a second, higher, proliferation threshold. If the IL-7R signaling strength is below its survival threshold, a cell may undergo apoptosis. When the signaling strength is above the survival threshold, but below the proliferation threshold, the cell survives but does not divide. Signaling strength above the proliferation threshold enables entry into cell cycle. Assuming that individual cell thresholds are log-normally distributed, we derive population-average rates for apoptosis and entry into cell cycle. We have analyzed the adiabatic change in homeostasis as thymic output decreases. With a parameter set representative of a healthy individual, the model predicts a unique equilibrium number of T cells. In a parameter range representative of persistent viral or bacterial infection, where naive T cell cycle progression is impaired, a decrease in thymic output may result in the collapse of the naive T cell repertoire.

Histone H2A is required for normal centromere function in Saccharomyces cerevisiae

PubMed Central

Pinto, Inés; Winston, Fred

2000-01-01

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G2–M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere–kinetochore function during chromosome segregation. PMID:10747028

Induction of S phase cell arrest and caspase activation by polysaccharide peptide isolated from Coriolus versicolor enhanced the cell cycle dependent activity and apoptotic cell death of doxorubicin and etoposide, but not cytarabine in HL-60 cells.

PubMed

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

2005-07-01

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

Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis

PubMed Central

Horlock-Roberts, Kathleen; Reaume, Chase; Dayer, Guillem; Ouellet, Christine; Cook, Nicholas

2017-01-01

ABSTRACT Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G1, S, and G2. Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G1 to late G2. Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle. IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission. PMID:28959734

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

PubMed

Qiu, Yueqin; Ma, Xianyong; Yang, Xuefen; Wang, Li; Jiang, Zongyong

2017-04-01

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.

Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

EPA Pesticide Factsheets

Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

Analysis of uncultured extremophilic snow algae by non-invasive single cell Raman spectroscopy

NASA Astrophysics Data System (ADS)

Beer, Thomas; Tanaka, Zuki; Netzter, Nathan; Rothschild, Lynn J.; Chen, Bin

2011-10-01

The study of life in extreme environments is a critical component of Astrobiology. But many of the so-called "extremophiles" are not readily cultivatable and therefore difficult to study under laboratory conditions. An example of such an extremophile is the snow alga Chlamydomonas cd. nivalis which expresses still unstudied secondary metabolites within its life cycle. In this paper, we present the first time the non-invasive single cell Raman spectroscopy of the life cycle dependent metabolite composition of C. nivalis. These secondary metabolites are likely related to the adaptation of C. nivalis to various stress factors. Normalized carotenoid Raman spectra intensities reveal characteristic ratio differences that allow identification of life cycle stages and putative secondary metabolites.

Regulation of apoptosis by low serum in cells of different stages of neoplastic progression: enhanced susceptibility after loss of a senescence gene and decreased susceptibility after loss of a tumor suppressor gene.

PubMed

Preston, G A; Lang, J E; Maronpot, R R; Barrett, J C

1994-08-01

A cell culture model system has been used to study the susceptibility of cells to apoptotic cell death during different stages of neoplastic progression. This system consists of normal diploid Syrian hamster embryo (SHE) cells, two preneoplastic cell lines [tumor suppressor stage I (sup +I) and non-tumor suppressor stage II (sup -II)], and hamster tumor cell lines. Stage I preneoplastic cells are nontumorigenic immortal clones that suppress tumorigenicity when hybridized to tumor cells, whereas stage II cells have lost the ability to suppress tumorigenicity in cell hybrids. We refer to these two types of preneoplastic cells as sup +I and sup -II, respectively. Neoplastic progression is generally associated with cellular alterations in growth factor responsiveness. Therefore, to study the regulation of apoptosis in the system described above, cells were cultured in low serum (0.2%) as a means of withdrawing growth factors. In low serum, normal SHE cells were quiescent (labeling index of 0.2%), with little cell death. The sup +I cells showed a relatively low labeling index (1.6%) but, in contrast to the normal cells, died at a high rate (55% cell loss after 48 h) by apoptosis, as evidenced by morphology, DNA fragmentation, and in situ end-labeling of fragmented DNA. The apoptotic cells did not go through a replicative cycle while in low serum, implying that apoptosis was initiated in the G0/G1 phase of the cell cycle. The sup -II cell line showed a high labeling index (40%) after 48 h, but cell growth was balanced by cell death that occurred at approximately the same rate. The cells died, however, predominantly by necrosis. The tumor cell lines continued to proliferate in low serum, with high labeling indices (ranging from 27% to 43%) and a low level of apoptotic or necrotic cell death. To determine the relative ability of these cells to survive in vivo, normal SHE cells, sup +I cells, and sup -II cells were injected s.c. into nude mice. At 5 or 21 days after injection, the normal SHE cells were readily retrieved from the mice and grew well in culture. In contrast, few sup +I cells were retrieved 5 days after injection and no viable cells were retrieved after 21 days. Sup -II cells were not retrieved at either the 5-day or 21-day harvest, and histological examinations of the sites of injection showed the presence of macrophages, eosinophils, and neutrophils, indicating an inflammatory response associated with necrotic cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats.

PubMed

Cuello-Carrión, F Darío; Troncoso, Mariana; Guiñazu, Elina; Valdez, Susana R; Fanelli, Mariel A; Ciocca, Daniel R; Kreimann, Erica L

2010-12-01

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

Radiosensitivity of Mammalian Cells

PubMed Central

Walters, R. A.; Petersen, D. F.

1968-01-01

Radiation effects on macromolecular synthesis essential for the Chinese hamster cell to traverse the life cycle and to divide have been investigated. Life-cycle analysis techniques employing inhibitors of macromolecular synthesis were used in determining the kinetics of cell growth for specific segments of the population following spontaneous recovery from radiation-induced division delay. The results indicated that recovery does not occur in the absence of functional protein synthesis. Under conditions which inhibit normal RNA and DNA synthesis, irradiated cells can recover the capacity to traverse the life cycle and to divide. The stability of mRNA species coding for proteins essential for division in irradiated cells was also measured. The mean functional lifetime of these mRNA species was 1 hr. The data demonstrate the existence of a specific segment of the population consisting of cells which have completed transcription related to division but not concomitant translation and which can recover from the radiation injury without synthesis of additional RNA. Thus, initial recovery of the ability to divide has an obligate requirement for protein synthesis but no corresponding requirement for nucleic acid synthesis during the period when original messenger remains intact. PMID:5753224

Inhibition of Estrogen-Induced Growth of Breast Cancer by Targeting Mitochondria Oxidants

DTIC Science & Technology

2010-04-01

acetylcysteine (NAC) and ebselen], inhibits estrogen induced expression of cell cycle genes as well as prevention of estrogen-induced growth of malignant breast...have completed proposed research in the original First Task (i) both antioxidants, N- acetylcysteine and ebselen, overexpression of ROS lowering genes...bioassay to test whether estrogen-induced conversion of normal cells to transformed cells is inhibited by treatment with N- acetylcysteine and

The ibrutinib B-cell proliferation inhibition is potentiated in vitro by dexamethasone: Application to chronic lymphocytic leukemia.

PubMed

Manzoni, Delphine; Catallo, Régine; Chebel, Amel; Baseggio, Lucile; Michallet, Anne-Sophie; Roualdes, Olivier; Magaud, Jean-Pierre; Salles, Gilles; Ffrench, Martine

2016-08-01

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

PubMed

Komaki, Yukako; Mariñas, Benito J; Plewa, Michael J

2014-10-07

Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of chromosomes may contribute to cancer induction and perhaps be involved in the induction of adverse pregnancy outcomes associated with long-term consumption of disinfected water. Here we present the first observation of the induction of hyperploidy by a class of DBPs.

NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature

PubMed Central

Al-Mayhani, M. Talal F.; Grenfell, Richard; Narita, Masashi; Piccirillo, Sara; Kenney-Herbert, Emma; Fawcett, James W.; Collins, V. Peter; Ichimura, Koichi; Watts, Colin

2011-01-01

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2− cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM. PMID:21798846

Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal.

PubMed

Jaafar Marican, Nur Hayati; Cruz-Migoni, Sara B; Borycki, Anne-Gaëlle

2016-06-14

Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Lactate dehydrogenase activity drives hair follicle stem cell activation

PubMed Central

Aimee, Flores; John, Schell; Abby, Krall; David, Jelinek; Matilde, Miranda; Melina, Grigorian; Daniel, Braas; White Andrew, C; Jessica, Zhou; Nick, Graham; Thomas, Graeber; Pankaj, Seth; Denis, Evseenko; Hilary, Coller; Jared, Rutter; Heather, Christofk; Lowry William, E

2017-01-01

Summary While normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli. PMID:28812580

Cell proliferation in normal epidermis

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

Weinstein, G.D.; McCullough, J.L.; Ross, P.

1984-06-01

A detailed examination of cell proliferation kinetics in normal human epidermis is presented. Using tritiated thymidine with autoradiographic techniques, proliferative and differentiated cell kinetics are defined and interrelated. The proliferative compartment of normal epidermis has a cell cycle duration (Tc) of 311 h derived from 3 components: the germinative labeling index (LI), the duration of DNA synthesis (ts), and the growth fraction (GF). The germinative LI is 2.7% +/- 1.2 and ts is 14 h, the latter obtained from a composite fraction of labeled mitoses curve obtained from 11 normal subjects. The GF obtained from the literature and from humanmore » skin xenografts to nude mice is estimated to be 60%. Normal-appearing epidermis from patients with psoriasis appears to have a higher proliferation rate. The mean LI is 4.2% +/- 0.9, approximately 50% greater than in normal epidermis. Absolute cell kinetic values for this tissue, however, cannot yet be calculated for lack of other information on ts and GF. A kinetic model for epidermal cell renewal in normal epidermis is described that interrelates the rate of birth/entry, transit, and/or loss of keratinocytes in the 3 epidermal compartments: proliferative, viable differentiated (stratum malpighii), and stratum corneum. Expected kinetic homeostasis in the epidermis is confirmed by the very similar ''turnover'' rates in each of the compartments that are, respectively, 1246, 1417, and 1490 cells/day/mm2 surface area. The mean epidermal turnover time of the entire tissue is 39 days. The Tc of 311 h in normal cells in 8-fold longer than the psoriatic Tc of 36 h and is necessary for understanding the hyperproliferative pathophysiologic process in psoriasis.« less

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

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

Mineo, C.; Murakami, Y.; Ishimi, Y.

1986-11-01

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33/sup 0/C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39/sup 0/C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39/sup 0/C for at least 10h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. Thismore » suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.« less

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

PubMed

Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

2015-08-01

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

Symbiotic Origin of Aging.

PubMed

Greenberg, Edward F; Vatolin, Sergei

2018-06-01

Normally aging cells are characterized by an unbalanced mitochondrial dynamic skewed toward punctate mitochondria. Genetic and pharmacological manipulation of mitochondrial fission/fusion cycles can contribute to both accelerated and decelerated cellular or organismal aging. In this work, we connect these experimental data with the symbiotic theory of mitochondrial origin to generate new insight into the evolutionary origin of aging. Mitochondria originated from autotrophic α-proteobacteria during an ancient endosymbiotic event early in eukaryote evolution. To expand beyond individual host cells, dividing α-proteobacteria initiated host cell lysis; apoptosis is a product of this original symbiont cell lytic exit program. Over the course of evolution, the host eukaryotic cell attenuated the harmful effect of symbiotic proto-mitochondria, and modern mitochondria are now functionally interdependent with eukaryotic cells; they retain their own circular genomes and independent replication timing. In nondividing differentiated or multipotent eukaryotic cells, intracellular mitochondria undergo repeated fission/fusion cycles, favoring fission as organisms age. The discordance between cellular quiescence and mitochondrial proliferation generates intracellular stress, eventually leading to a gradual decline in host cell performance and age-related pathology. Hence, aging evolved from a conflict between maintenance of a quiescent, nonproliferative state and the evolutionarily conserved propagation program driving the life cycle of former symbiotic organisms: mitochondria.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

PubMed

Machado, Kátia da Conceição; Sousa, Lívia Queiroz de; Lima, Daisy Jereissati Barbosa; Soares, Bruno Marques; Cavalcanti, Bruno Coêlho; Maranhão, Sarah Sant'Anna; Noronha, Janaina da Costa de; Rodrigues, Domingos de Jesus; Militão, Gardenia Carmen Gadelha; Chaves, Mariana Helena; Vieira-Júnior, Gerardo Magela; Pessoa, Cláudia; Moraes, Manoel Odorico de; Sousa, João Marcelo de Castro E; Melo-Cavalcante, Ana Amélia de Carvalho; Ferreira, Paulo Michel Pinheiro

2018-03-15

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC 50 values ranging from 0.15 (leukemia) to 7.35 (larynx) μM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC 50: 10.88 μM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC 50 : 7.5 μM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Embryological outcomes in cycles with human oocytes containing large tubular smooth endoplasmic reticulum clusters after conventional in vitro fertilization.

PubMed

Itoi, Fumiaki; Asano, Yukiko; Shimizu, Masashi; Honnma, Hiroyuki; Murata, Yasutaka

2016-01-01

There have been no studies analyzing the effect of large aggregates of tubular smooth endoplasmic reticulum (aSERT) after conventional in vitro fertilization (cIVF). The aim of this study was to investigate whether aSERT can be identified after cIVF and the association between the embryological outcomes of oocytes in cycles with aSERT. This is a retrospective study examining embryological data from cIVF cycles showing the presence of aSERT in oocytes 5-6 h after cIVF. To evaluate embryo quality, cIVF cycles with at least one aSERT-metaphase II (MII) oocyte observed (cycles with aSERT) were compared to cycles with normal-MII oocytes (control cycles). Among the 4098 MII oocytes observed in 579 cycles, aSERT was detected in 100 MII oocytes in 51 cycles (8.8%). The fertilization rate, the rate of embryo development on day 3 and day 5-6 did not significantly differ between cycles with aSERT and control group. However, aSERT-MII oocytes had lower rates for both blastocysts and good quality blastocysts (p < 0.05). aSERT can be detected in the cytoplasm by removing the cumulus cell 5 h after cIVF. However, aSERT-MII oocytes do not affect other normal-MII oocytes in cycles with aSERT.

The Septins Function in G1 Pathways that Influence the Pattern of Cell Growth in Budding Yeast

PubMed Central

Egelhofer, Thea A.; Villén, Judit; McCusker, Derek; Gygi, Steven P.; Kellogg, Douglas R.

2008-01-01

The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation. PMID:18431499

Peptide promotes overcoming of the division limit in human somatic cell.

PubMed

Khavinson, V Kh; Bondarev, I E; Butyugov, A A; Smirnova, T D

2004-05-01

We previously showed that treatment of normal human diploid cells with Epithalon (Ala-Glu-Asp-Gly) induced expression of telomerase catalytic subunit, its enzymatic activity, and elongation of telomeres. Here we studied the effect of this peptide on proliferative potential of human fetal fibroblasts. Primary pulmonary fibroblasts derived from a 24-week fetus lost the proliferative potential at the 34th passage. The mean size of telomeres in these cells was appreciably lower than during early passages (passage 10). Addition of Epithalon to aging cells in culture induced elongation of telomeres to the size comparable to their length during early passages. Peptide-treated cells with elongated telomeres made 10 extra divisions (44 passages) in comparison with the control and continued dividing. Hence, Epithalon prolonged the vital cycle of normal human cells due to overcoming the Heyflick limit.

[Effects of Rhizoma kaempferiae volatile oil on tumor growth and cell cycle of MKN-45 human gastric cancer cells orthotopically transplanted in nude mice].

PubMed

Xiao, Yan; Wei, Pin-Kang; Li, Jun; Shi, Jun; Yu, Zhi-Hong; Lin, Hui-Ming

2006-07-01

To evaluate the effects of Rhizoma kaempferiae volatile oil on tumor growth and cell cycle of MKN-45 human gastric cancer cells orthotopically transplanted in nude mice. One hundred and five nude mice orthotopically transplanted with MKN-45 human gastric cancer cells were randomly divided into seven groups: untreated group, normal saline-treated group, dissolvant-treated group, cyclophosphamide (CTX)-treated group and high-, medium-, and low-dose Rhizoma kaempferiae volatile oil-treated groups. Corresponding interventions were implemented in each group except the untreated group. The antitumor effects in vivo were evaluated. Cell cycle distribution and apoptosis of MKN-45 human gastric cancer cells were determined by using flow cytometry (FCM). The ultrastructure of MKN-45 gastric cancer cells was observed by a transmission electron microscope. In the high-, medium-, and low-dose Rhizoma kaempferiae volatile oil-treated groups, the growth inhibition rates of gastric cancer were 57.2%, 28.0% and 5.0% respectively, and the gastric cancer cells were arrested at G(0)/G(1) phase. This antitumor effect was dose-dependent. The apoptotic cells occurred more frequently in the high-dose Rhizoma kaempferiae volatile oil-treated group and the CTX-treated group than those in the medium- and low-dose Rhizoma kaempferiae volatile oil-treated groups. The Rhizoma kaempferiae volatile oil is an effective composition for growth inhibition of gastric cancer, and its mechanism may be related to regulating the cell cycle and inducing apoptosis.

MicroRNA inhibition fine-tunes and provides robustness to the restriction point switch of the cell cycle.

PubMed

Del Rosario, Ricardo C H; Damasco, Joseph Ray Clarence G; Aguda, Baltazar D

2016-09-09

The restriction point marks a switch in G1 from growth factor-dependent to growth factor-independent progression of the cell cycle. The proper regulation of this switch is important for normal cell processes; aberrations could result in a number of diseases such as cancer, neurodegenerative disorders, stroke and myocardial infarction. To further understand the regulation of the restriction point, we extended a mathematical model of the Rb-E2F pathway to include members of the microRNA cluster miR-17-92. Our mathematical analysis shows that microRNAs play an essential role in fine-tuning and providing robustness to the switch. We also demonstrate how microRNA regulation can steer cells in or out of cancer states.

MicroRNA inhibition fine-tunes and provides robustness to the restriction point switch of the cell cycle

PubMed Central

del Rosario, Ricardo C. H.; Damasco, Joseph Ray Clarence G.; Aguda, Baltazar D.

2016-01-01

The restriction point marks a switch in G1 from growth factor-dependent to growth factor-independent progression of the cell cycle. The proper regulation of this switch is important for normal cell processes; aberrations could result in a number of diseases such as cancer, neurodegenerative disorders, stroke and myocardial infarction. To further understand the regulation of the restriction point, we extended a mathematical model of the Rb-E2F pathway to include members of the microRNA cluster miR-17-92. Our mathematical analysis shows that microRNAs play an essential role in fine-tuning and providing robustness to the switch. We also demonstrate how microRNA regulation can steer cells in or out of cancer states. PMID:27610602

C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

PubMed Central

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. PMID:25483090

[Immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from patients with gastroesophageal reflux disease].

PubMed

Koyama, Shigeki; Nishiyama, Yorihiro; Ishizuka, Izumi

2007-05-01

We performed an immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from the patients with gastroesophageal reflux disease (GERD) using angled -biopsy forceps. Inflammatory cell accumulation into the lamina propria was detected even in patients with modified Los Angeles (LA) system grades N or M. In grade N or M patients with no changes in the epithelium, the area of p21, p27 and Ki67 positive cells was expanded compared to normal mucosa. The area of p21, p27 and Ki67 positive cells tended to expand upward in the epithelium with GERD severity based on the LA classification grading. These indicate that inflammatory cell infiltration into the lamina propria is initial histological change of GERD.

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

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.

GROWTH REGULATION IN RSV INFECTED CHECKEN EMBRYO FIBROBLASTS: THE ROLE OF THE src GENE

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

Parry, G.; Bartholomew, J.C.; Bissell, M.J.

1980-03-01

The relationship between growth regulation and cell transformation has been studied in many cultured cell lines transformed by a range of oncogenic agents. The main conclusion derived from these investigations is that the nature of the growth regulatory lesion in transformed cells is a function of the agent used to induce transformation. For example, when 3T3 fibroblasts are rendered stationary by serum deprivation, normal cells accumulate in G{sub 1} but SV40 transformed cells are arrested at all stages of the cell cycle. In contrast, 3T3 cells transformed with Rous sarcoma virus B77, accumulate in G{sub 1} upon serum deprivation. Thismore » is also true when mouse sarcoma virus (MSV) is used as the transforming agent. MSV-transformed cells accumulate in G{sub 1}, just as do normal cells. In this letter we report a detailed study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in the process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. Two principal findings have emerged: (a) the stationary phase of the cell cycle (G{sub 1}) in chick embryo fibroblasts has two distinct compartments, (for simplicity referred to as G{sub 1} and G{sub 0} states), (b) when rendered stationary at 41.5{sup o} by serum deprivation, normal cells enter a G{sub 0}-like state, but cells infected with the ts-mutant occupy a G{sub 1} state, even though a known src gene product, a kinase, should be inactive at this temperature. The possibility is discussed that viral factors other than the active src protein kinase influence growth control.« less

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

PubMed Central

McAllister, Jan M.; Modi, Bhavi; Miller, Bruce A.; Biegler, Jessica; Bruggeman, Richard; Legro, Richard S.; Strauss, Jerome F.

2014-01-01

Polycystic ovary syndrome (PCOS), characterized by increased ovarian androgen biosynthesis, anovulation, and infertility, affects 5–7% of reproductive-age women. Genome-wide association studies identified PCOS candidate loci that were replicated in subsequent reports, including DENND1A, which encodes a protein associated with clathrin-coated pits where cell-surface receptors reside. However, these studies provided no information about functional roles for DENND1A in the pathogenesis of PCOS. DENND1A protein was located in the cytoplasm as well as nuclei of theca cells, suggesting a possible role in gene regulation. DENND1A immunostaining was more intense in the theca of PCOS ovaries. Using theca cells isolated and propagated from normal cycling and PCOS women, we found that DENND1A variant 2 (DENND1A.V2) protein and mRNA levels are increased in PCOS theca cells. Exosomal DENND1A.V2 RNA was significantly elevated in urine from PCOS women compared with normal cycling women. Forced overexpression of DENND1A.V2 in normal theca cells resulted in a PCOS phenotype of augmented CYP17A1 and CYP11A1 gene transcription, mRNA abundance, and androgen biosynthesis. Knock-down of DENND1A.V2 in PCOS theca cells reduced androgen biosynthesis and CYP17A1 and CYP11A1 gene transcription. An IgG specific to DENND1A.V2 also reduced androgen biosynthesis and CYP17 and CYP11A1 mRNA when added to the medium of cultured PCOS theca cells. We conclude that the PCOS candidate gene, DENND1A, plays a key role in the hyperandrogenemia associated with PCOS. These observations have both diagnostic and therapeutic implications for this common disorder. PMID:24706793

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

PubMed

Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p < .05 to p < .001). The antiproliferative effects of I3C were in association with programed cell death. I3C downregulated BCL2 and upregulated FasR in THP-1 cells (p < .05 to p < .001). G1 cell cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p < .05 to p < .001), while CDK2 was downregulated upon I3C treatment (p < .01 to p < .001). I3C could exert its antileukemic effects through AhR activation which is associated with programed cell death and G1 cell cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells1

PubMed Central

Jaiswal, Aruna S; Panda, Harekrushna; Pampo, Christine A; Siemann, Dietmar W; Gairola, C Gary; Hromas, Robert; Narayan, Satya

2013-01-01

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model. PMID:23555190

Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice.

PubMed

Liu, Nan; Wang, Lin-Hui; Guo, Ling-Ling; Wang, Guo-Qing; Zhou, Xi-Ping; Jiang, Yan; Shang, Jing; Murao, Koji; Chen, Jing-Wei; Fu, Wen-Qing; Zhang, Guo-Xing

2013-01-01

Solid evidence has demonstrated that psychoemotional stress induced alteration of hair cycle through neuropeptide substance P (SP) mediated immune response, the role of reactive oxygen species (ROS) in brain-skin-axis regulation system remains unknown. The present study aims to investigate possible mechanisms of ROS in regulation of SP-mast cell signal pathway in chronic restraint stress (CRS, a model of chronic psychoemotional stress) which induced abnormal of hair cycle. Our results have demonstrated that CRS actually altered hair cycle by inhibiting hair follicle growth in vivo, prolonging the telogen stage and delaying subsequent anagen and catagen stage. Up-regulation of SP protein expression in cutaneous peripheral nerve fibers and activation of mast cell were observed accompanied with increase of lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in CRS mice skin. In addition, SP receptor antagonist (RP67580) reduced mast cell activations and lipid peroxidation levels as well as increased GSH-Px activity and normalized hair cycle. Furthermore, antioxidant Tempol (a free radical scavenger) also restored hair cycle, reduced SP protein expression and mast cell activation. Our study provides the first solid evidence for how ROS play a role in regulation of psychoemotional stress induced SP-Mast cell pathway which may provide a convincing rationale for antioxidant application in clinical treatment with psychological stress induced hair loss.

[Circadian rhythm variation of the clock genes Per1 and cell cycle related genes in different stages of carcinogenesis of buccal mucosa in animal model].

PubMed

Tan, Xuemei; Ye, Hua; Yang, Kai; Chen, Dan; Tang, Hong

2015-07-01

To investigate the expression and circadian rhythm variation of biological clock gene Per1 and cell cycle genes p53, CyclinD1, cyclin-dependent kinases (CDK1), CyclinB1 in different stages of carcinogenesis in buccal mucosa and its relationship with the development of buccal mucosa carcinoma. Ninety golden hamsters were housed under 12 hours light-12 hours dark cycles, and the model of buccal squamous cell carcinoma was established by using the dimethylbenzanthracene (DMBA) to smear the golden hamster buccal mucosa. Before the DMBA was used and after DMBA was used 6 weeks and 14 weeks respectively, the golden hamsters were sacrificed at 6 different time points (5 rats per time point) within 24 hour, including 4, 8, 12, 16, 20 and 24 hour after lights onset (HALO), and the normal buccal mucosa, precancerous lesions and cancer tissue were obtained, respectively. HE stained sections were prepared to observe the canceration of each tissue. Real time RT-PCR was used to detect the mRNA expression of Per1, p53, CyclinD1, CDK1 and CyclinB1, and a cosine analysis method was applied to determine the circadian rhythm variation of Per1, p53, CyclinD1, CDK1 and CyclinB1 mRNA expression, which were characterized by median, amplitude and acrophase. The expression of Per1, p53, CDK1 and CyclinD1 mRNA in 6 different time points within 24 hours in the tissues of three different stages of carcinogenesis had circadian rhythm, respectively. However, the CyclinB1 mRNA was expressed with circadian rhythm just in normal and cancer tissue (P < 0.05), while in precancerous lesions the circadian rhythm was in disorder (P > 0.05). As the development of carcinoma, the median of Per1 and p53 mRNA expression were significantly decreased (P < 0.05), yet the median of CDK1, CyclinB1 and CyclinD1 mRNA expression were significantly increased (P < 0.05). The amplitude of Per1, p53 and CyclinD1 mRNA expression was significantly decreased as the development of carcinoma (P < 0.05), however the amplitude of CDK1 mRNA expression was significantly increased (P < 0.05). In addition, there was no significant difference in the amplitude of CyclinB1 mRNA expression. The time that the peak expression value of Per1 and CDK1 mRNA appeared (Acrophase) in precancerous lesions was remarkably earlier than that in normal tissues, but the acrophase of p53 and CyclinD1 mRNA expression was remarkably delayed. Moreover, the acrophase of CDK1 and CyclinB1 mRNA expression in cancer tissues was obviously earlier than that in normal tissues, yet there was no significant variation in acrophase of Per1, p53, CyclinD1 mRNA expression between normal tissues and cancer tissues. The circadian rhythm of clock gene Per1 and cell cycle genes p53, CyclinD1, CDK1, CyclinB1 expression remarkably varied with the occurrence and development of carcinoma. Further research into the interaction between circadian and cell cycle of two cycle activity and relationship with the carcinogenesis may providenew ideas and methods of individual treatment and the mechanism of carcinogenesis.

Cell cycles and proliferation patterns in Haematococcus pluvialis

NASA Astrophysics Data System (ADS)

Zhang, Chunhui; Liu, Jianguo; Zhang, Litao

2017-09-01

Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation; far less attention has been paid to cell cycles and proliferation patterns. The purpose of this study was to clarify cell cycles and proliferation patterns in H. pluvialis microscopically using a camera and video recorder system. The complicated life history of H. pluvialis can be divided into two stages: the motile stage and the non-motile stage. All the cells can be classified into forms as follows: motile cell, nonmotile cell, zoospore and aplanospore. The main cell proliferation, both in the motile phase and non-motile phase in H. pluvialis, is by asexual reproduction. Under normal growth conditions, a motile cell usually produces two, sometimes four, and exceptionally eight zoospores. Under unfavorable conditions, the motile cell loses its flagella and transforms into a non-motile cell, and the non-motile cell usually produces 2, 4 or 8 aplanospores, and occasionally 20-32 aplanospores, which further develop into non-motile cells. Under suitable conditions, the non-motile cell is also able to release zoospores. The larger non-motile cells produce more than 16 zoospores, and the smaller ones produce 4 or 8 zoospores. Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase. There is, as yet, no convincing direct evidence for sexual reproduction.

Molecular characterization of immortalized normal and dysplastic oral cell lines.

PubMed

Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

2015-05-01

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

North- and South-Bicyclo[3.1.0]Hexene Nucleosides: The Effect of Ring Planarity on Anti-HIV Activity | Center for Cancer Research

Cancer.gov

The picture shows the locked north (blue) and south (red) bicyclo[3.1.0]hexane nucleosides in the normal pseudorotational cycle, and the corresponding shift to a smaller cycle (nmax=7°) caused by the insertion of a double bond. The former nucleosides are inactive, while the flattening of the embedded cyclopentene ring provides active compounds against HOS cells infected with

Targeted Zinc Delivery: A Novel Treatment for Prostate Cancer

DTIC Science & Technology

2010-06-01

aconitase, which normally functions to oxidize citrate during the Krebs cycle . Because citrate is a principle component of seminal fluid, prostate...tissue, likely due to the metabolic effects of zinc in the Krebs cycle . That is, because zinc inhibits m- aconitase, loss of zinc allows for greater...secretory cells do not complete the oxidation of citrate in the mitochondria and the zinc-mediated inhibition of m-aconitase is crucial for the

The Ubiquitin-Conjugating Enzyme E2-EPF Is Overexpressed in Primary Breast Cancer and Modulates Sensitivity to Topoisomerase II Inhibition1

PubMed Central

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-01-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER- MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G2/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G2 checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIα protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness. PMID:17710163

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

PubMed

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-07-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

Simulated microgravity, Mars gravity, and 2g hypergravity affect cell cycle regulation, ribosome biogenesis, and epigenetics in Arabidopsis cell cultures.

PubMed

Kamal, Khaled Y; Herranz, Raúl; van Loon, Jack J W A; Medina, F Javier

2018-04-23

Gravity is the only component of Earth environment that remained constant throughout the entire process of biological evolution. However, it is still unclear how gravity affects plant growth and development. In this study, an in vitro cell culture of Arabidopsis thaliana was exposed to different altered gravity conditions, namely simulated reduced gravity (simulated microgravity, simulated Mars gravity) and hypergravity (2g), to study changes in cell proliferation, cell growth, and epigenetics. The effects after 3, 14, and 24-hours of exposure were evaluated. The most relevant alterations were found in the 24-hour treatment, being more significant for simulated reduced gravity than hypergravity. Cell proliferation and growth were uncoupled under simulated reduced gravity, similarly, as found in meristematic cells from seedlings grown in real or simulated microgravity. The distribution of cell cycle phases was changed, as well as the levels and gene transcription of the tested cell cycle regulators. Ribosome biogenesis was decreased, according to levels and gene transcription of nucleolar proteins and the number of inactive nucleoli. Furthermore, we found alterations in the epigenetic modifications of chromatin. These results show that altered gravity effects include a serious disturbance of cell proliferation and growth, which are cellular functions essential for normal plant development.

Cancer prevention, the need to preserve the integrity of the genome at all cost.

PubMed

Okafor, M T; Nwagha, T U; Anusiem, C; Okoli, U A; Nubila, N I; Al-Alloosh, F; Udenyia, I J

2018-05-01

The entire genetic information carried by an organism makes up its genome. Genes have a diverse number of functions. They code different proteins for normal proliferation of cells. However, changes in the base sequence of genes affect their protein by-products which act as messengers for normal cellular functions such as proliferation and repairs. Salient processes for maintaining the integrity of the genome are hinged on intricate mechanisms put in place for the evolution to tackle genomic stresses. To discuss how cells sense and repair damage to their deoxyribonucleic acid (DNA) as well as to highlight how defects in the genes involved in DNA repair contribute to cancer development. Methodology: Online searches on the following databases such as Google Scholar, PubMed, Biomed Central, and SciELO were done. Attempt was made to review articles with keywords such as cancer, cell cycle, tumor suppressor genes, and DNA repair. The cell cycle, tumor suppression genes, DNA repair mechanism, as well as their contribution to cancer development, were discussed and reviewed. Knowledge on how cells detect and repair DNA damage through an array of mechanisms should allay our anxiety as regards cancer development. More studies on DNA damage detection and repair processes are important toward a holistic approach to cancer treatment.

The adverse effects of high fat induced obesity on female reproductive cycle and hormones

NASA Astrophysics Data System (ADS)

Donthireddy, Laxminarasimha Reddy

The prevalence of obesity, an established risk and progression factor for abnormal reproductive cycle and tissue damage in female mice. It leads to earlier puberty, menarche in young females and infertility. There are extensive range of consequences of obesity which includes type-2 diabetes, cardiovascular disease and insulin resistance. Obesity is the interaction between dietary intake, genes, life style and environment. The interplay of hormones estrogen, insulin, and leptin is well known on energy homeostasis and reproduction. The aim of this study is to determine the effect of high fat induced obesity on reproductive cycles and its hormonal abnormalities on mice model. Two week, 3 month and 8 month long normal (WT) and very high fat diet (VHFD) diet course is followed. When mice are fed with very high fat diet, there is a drastic increase in weight within the first week later. There was a significant (p<0.001) increase in leptin levels in 6 month VHFD treated animals. 2 week, 3 month and 6 month time interval pap smear test results showed number of cells, length of estrous cycle and phases of the estrous cycle changes with VHFD mice(n=30) compared to normal diet mice(n=10). These results also indicate that the changes in the reproductive cycles in VHFD treated female mice could be due to the changes in hormones. Histo-pathological analyses of kidney, ovary, liver, pancreas, heart and lungs showed remarkable changes in some tissue on exposure to very high fat. Highly deposited fat packets observed surrounding the hepatocytes and nerve cells.

Qualification testing of general electric 50 Ah nickel-cadmium cells with new separator and new positive plate processing

NASA Astrophysics Data System (ADS)

Morrow, G. W.

1986-09-01

Forty-two 50 Ah aerospace nickel-cadmium cells were delivered to Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985, for the purpose of evaluating and qualifying a new nylon separator material Pellon 2536, and the new GE Positive Plate Nickel Attack Control Passivation process. Testing began in May, 1985, at the Naval Weapons Support Center (NWSC) in Crane, Indiana with standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985, with approximately 1200 LEO cycles complete at this writting. Early test results show that cells with positive plate passivation exhibit higher than normal charge voltage characteristics. Other aspects of performance were nominal.

Qualification Testing of General Electric 50 Ah Nickel-Cadmium Cells with New Separator and New Positive Plate Processing

NASA Technical Reports Server (NTRS)

Morrow, G. W.

1986-01-01

Forty-two 50 Ah aerospace nickel-cadmium cells were delivered to Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985, for the purpose of evaluating and qualifying a new nylon separator material Pellon 2536, and the new GE Positive Plate Nickel Attack Control Passivation process. Testing began in May, 1985, at the Naval Weapons Support Center (NWSC) in Crane, Indiana with standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985, with approximately 1200 LEO cycles complete at this writting. Early test results show that cells with positive plate passivation exhibit higher than normal charge voltage characteristics. Other aspects of performance were nominal.

A Novel, Non-Apoptotic Role for Scythe/BAT3: A Functional Switch between the Pro- and Anti-Proliferative Roles of p21 during the Cell Cycle

PubMed Central

Yong, Sheila T.; Wang, Xiao-Fan

2012-01-01

Background Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. Methods/Principal Findings Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. Conclusion: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle. PMID:22761665

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

NASA Astrophysics Data System (ADS)

Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

2009-12-01

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

Methionine sulfoxide reductase A regulates cell growth through the p53-p21 pathway

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

Choi, Seung Hee; Kim, Hwa-Young, E-mail: hykim@ynu.ac.kr

2011-12-09

Highlights: Black-Right-Pointing-Pointer Down-regulation of MsrA inhibits normal cell proliferation. Black-Right-Pointing-Pointer MsrA deficiency leads to an increase in p21 by enhanced p53 acetylation. Black-Right-Pointing-Pointer Down-regulation of MsrA causes cell cycle arrest at the G{sub 2}/M stage. Black-Right-Pointing-Pointer MsrA is a regulator of cell growth that mediates the p53-p21 pathway. -- Abstract: MsrA is an oxidoreductase that catalyzes the stereospecific reduction of methionine-S-sulfoxide to methionine. Although MsrA is well-characterized as an antioxidant and has been implicated in the aging process and cellular senescence, its roles in cell proliferation are poorly understood. Here, we report a critical role of MsrA in normal cellmore » proliferation and describe the regulation mechanism of cell growth by this protein. Down-regulation of MsrA inhibited cell proliferation, but MsrA overexpression did not promote it. MsrA deficiency led to an increase in p21, a major cyclin-dependent kinase inhibitor, thereby causing cell cycle arrest at the G{sub 2}/M stage. While protein levels of p53 were not altered upon MsrA deficiency, its acetylation level was significantly elevated, which subsequently activated p21 transcription. The data suggest that MsrA is a regulator of cell growth that mediates the p53-p21 pathway.« less

Gene expression profiling: cell cycle deregulation and aneuploidy do not cause breast cancer formation in WAP-SVT/t transgenic animals.

PubMed

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

2005-05-01

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

Prevention of radiation-induced salivary gland dysfunction utilizing a CDK inhibitor in a mouse model.

PubMed

Martin, Katie L; Hill, Grace A; Klein, Rob R; Arnett, Deborah G; Burd, Randy; Limesand, Kirsten H

2012-01-01

Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues. Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G(2)/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G(2)/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output. These studies suggest that induction of transient G(2)/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.

E2F activators signal and maintain centrosome amplification in breast cancer cells.

PubMed

Lee, Mi-Young; Moreno, Carlos S; Saavedra, Harold I

2014-07-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2(+) cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2(+) cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2(+) breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.

The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. III. Kinetics of cell proliferation in the external granular layer.

PubMed

Lauder, J M

1977-04-22

The effects of early hypo- and hyperthyroidism on the rates of cell acquisition and proliferation have been studied in the external granular layer (EGL) of the developing rat cerebellar cortex at 10 days of age using quantitative autoradiographic methods. Both altered thyroid states reduce the rate of cell acquisition in the EGL, but appear to do so for different reasons. Hyperthyroidism shortens the average length of the cell cycle by decreasing the duration of the pre-DNA synthetic phase (G1), indicating that excess thyroxine may exert a direct effect on the EGL. This action involves the early onset of neuronal differentiation (cessation of proliferation)46 which presumably leads to the observed decrease in the rate of cell acquisition (increased doubling time). Such differentiating cells do not, however, leave the proliferative zone or the EGL prematurely, resulting in a reduced labeling index, mitotic index, and growth fraction as non-dividing cells dilute the proliferating cell population. Hypothyroidism, on the other hand, leads to no significant change in the length of the cell cycle or in the mitotic index, but causes a decreased labeling index and growth fraction, as well as a reduced rate of cell acquisition (increased doubling time). No significant change in the amount of cell death in the EGL could be found to explain this apparent discrepancy between the rate of cell proliferation (cell cycle length) and cell acqusiition. The answer to this puzzle appears to lie in the mitotic index, which is not affected to the same extent as the labeling index, although it is also slightly reduced. If cells were to remain longer in mitosis, this could result in a decreased labeling index and growth fraction but nearly normal mitotic index and cell cycle length (as measured using the % labeled mitoses method), since those cells dropping out of the cycling population would be counted as mitoses...

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

STAT proteins: from normal control of cellular events to tumorigenesis.

PubMed

Calò, Valentina; Migliavacca, Manuela; Bazan, Viviana; Macaluso, Marcella; Buscemi, Maria; Gebbia, Nicola; Russo, Antonio

2003-11-01

Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis.

Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

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

Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity wasmore » assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing gammaH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2mug/mL], Trinovin [10mug/mL], and Prostate Rx [50 mug/mL]). However, both Trinovin (10mug/mL) and Prostate Rx (6mug/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.« less

Ionizing radiation and cell cycle progression in ataxia telangiectasia

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

Beamish, H.; Khanna, K.K.; Lavin, M.F.

1994-04-01

Exposure of mammalian cells to ionizing radiation causes delay in normal progress through the cell cycle at a number of different checkpoints. Abnormalities in these checkpoints have been described for ataxia telangiectasia cells after irradiation. In this report we show that these abnormalities occur at different phases in the cell cycle in several ataxia telangiectasia lymphoblastoid cells. Ataxia telangiectasia cells, synchronized in late G{sub 1} phase with either mimosine or aphidicolin and exposed to radiation, showed a reduced delay in entering S phase compared to irradiated control cells. Failure to exhibit G{sub 1}-phase delay in ataxia telangiectasia cells is accompaniedmore » by a reduced ability of radiation to activate the product of the tumor suppressor gene p53, a protein involved in G{sub 1}/S-phase delay. When the progress of irradiated G{sub 1}-phase cells was followed into the subsequent G{sub 2} and G{sub 1} phases ataxia telangiectasia cells showed a more pronounced accumulation in G{sub 2} phase than control cells. When cells were irradiated in S phase and extent of delay was more evident in G{sub 2} phase and ataxia telangiectasia cells were delayed to a greater extent. These results suggest that the lack of initial delay in both G{sub 1} and S phases to the radiosensitivity observed in this syndrome. 26 refs., 3 figs., 2 tabs.« less

What Is Breast in the Bone?

PubMed

Shemanko, Carrie S; Cong, Yingying; Forsyth, Amanda

2016-10-22

The normal developmental program that prolactin generates in the mammary gland is usurped in the cancerous process and can be used out of its normal cellular context at a site of secondary metastasis. Prolactin is a pleiotropic peptide hormone and cytokine that is secreted from the pituitary gland, as well as from normal and cancerous breast cells. Experimental and epidemiologic data suggest that prolactin is associated with mammary gland development, and also the increased risk of breast tumors and metastatic disease in postmenopausal women. Breast cancer spreads to the bone in approximately 70% of cases with advanced breast cancer. Despite treatment, new bone metastases will still occur in 30%-50% of patients. Only 20% of patients with bone metastases survive five years after the diagnosis of bone metastasis. The breast cancer cells in the bone microenvironment release soluble factors that engage osteoclasts and/or osteoblasts and result in bone breakdown. The breakdown of the bone matrix, in turn, enhances the proliferation of the cancer cells, creating a vicious cycle. Recently, it was shown that prolactin accelerated the breast cancer cell-mediated osteoclast differentiation and bone breakdown by the regulation of breast cancer-secreted proteins. Interestingly, prolactin has the potential to affect multiple proteins that are involved in both breast development and likely bone metastasis, as well. Prolactin has normal bone homeostatic roles and, combined with the natural "recycling" of proteins in different tissues that can be used for breast development and function, or in bone function, increases the impact of prolactin signaling in breast cancer bone metastases. Thus, this review will focus on the role of prolactin in breast development, bone homeostasis and in breast cancer to bone metastases, covering the molecular aspects of the vicious cycle.

Effect of Temporal Pattern of Radiation in Intensity Modulated Radiotherapy on Cell Cycle Progression and Apoptosis of ACHN Renal Cell Carcinoma Cell Line.

PubMed

Khorramizadeh, Maryam; Saberi, Alihossein; Tahmasebi-Birgani, Mohammadjavad; Shokrani, Parvaneh; Amouhedari, Alireza

The existence of a hypersensitive radiation response to doses below 1 Gy is well established for many normal and tumor cell lines. The aim of this study was to ascertain the impact of temporal pattern modeling IMRT on survival, cell cycle and apoptosis of human RCC cell line ACHN, so as to provide radiobiological basis for optimizing IMRT plans for this disease. The ACHN renal cell carcinoma cell line was used in this study. Impact of the triangle, V, small-large or large-small temporal patterns in the presence and absence of threshold dose of hyper-radiosensitivity at the beginning of patterns were studied using soft agarclonogenic assays. Cell cycle and apoptosis analysis were performed after irradiation with the temporal patterns. For triangle and small-large dose sequences, survival fraction was significantly reduced after irradiation with or without threshold dose of hyper-radiosensitivity at the beginning of the patterns. In all of the dose patterns, cell cycle distributions and the percentage of apoptotic cells at 24 h after irradiation with or without priming dose of hyper-radiosensitivity showed no significant difference. However, apoptotic cells were increased when beams with the smallest dose applied at the beginning of dose pattern like triangle and small-large dose sequence. These data show that the biologic effects of single fraction may differ in clinical settings depending on the size and sequence of the partial fractions. Doses at the beginning but not at the end of sequences may change cytotoxicity effects of radiation.

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

PubMed Central

Maningat, Patricia D.; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L.; Bray, Molly S.

2011-01-01

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7–10 days. PMID:21205870

High expression of Polycomb group protein EZH2 predicts poor survival in salivary gland adenoid cystic carcinoma.

PubMed

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

2008-06-01

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

Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline

PubMed Central

Chi, Woo; Wu, Eleanor; Morgan, Bruce A.

2013-01-01

Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss. PMID:23487317

Evaluation of advanced glycation end-products in diabetic and inherited canine cataracts.

PubMed

Bras, I Dineli; Colitz, Carmen M H; Kusewitt, Donna F; Chandler, Heather; Lu, Ping; Gemensky-Metzler, Anne J; Wilkie, David A

2007-02-01

The receptor for advanced glycation end-products (RAGE) increases in the human cataract and should correlate with increased DNA damage and proliferation of lens epithelial cells (LECs). The purpose of this study was to measure and immunolocalize RAGE in normal and cataractous canine LECs, and to determine whether there was a correlation between RAGE and DNA damage (gadd45), cell-cycle regulation (p21), and LEC proliferation (proliferating cell nuclear antigen, PCNA). Thirty-two anterior lens capsules from 22 dogs that underwent cataract surgery and 10 lenses from dogs with normal eyes were evaluated. Eleven of the cataractous lenses were from diabetic patients (n=16), and eleven were from patients with inherited cataracts (n=16). Standard immunohistochemical staining was performed using antibodies against RAGE, gadd45, p21, PCNA, alpha-smooth muscle actin, and TGF-beta. Immunostaining intensity for each antibody was given a score of 0-4+. Standard Western blot analysis on normal and cataractous lens capsules was performed using the same antibodies as in the immunohistochemical staining. Comparisons were also made based on age and sex. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for RAGE. There was an increase in RAGE expression with age in normal LECs, but no significant difference was seen when normal adult LECs were compared to cataractous LECs. The stage of the cataract and the presence of LIU were not associated with a significant increase in RAGE expression. There was no age-dependent difference in the normal lenses for gadd45, p21, or PCNA. Significant up-regulation of p21 (P < 0.05) and PCNA (P < 0.05) was seen in diabetic cataracts compared to inherited cataracts. RAGE and PCNA expression did not increase with cataractogenesis, possibly due to overexpression associated with normal aging and constant exposure to oxidative stress from sunlight-related ultraviolet irradiation, respectively. However, p21 and PCNA increased in diabetic cataractogenesis suggesting cell cycle and proliferation dysregulation. This may be related to the rapid onset in this type of cataract compared with the more chronic and slower-to-develop inherited cataracts.

Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

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

Wang, Ruoxing; Guo, Yan-Lin, E-mail: yanlin.guo@usm.edu

2012-10-01

Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remainsmore » unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: Black-Right-Pointing-Pointer Inhibition of Cdks slows down mESCs proliferation. Black-Right-Pointing-Pointer mESCs display remarkable recovery capacity from short-term cell cycle interruption. Black-Right-Pointing-Pointer Short-term cell cycle interruption does not compromise mESC self-renewal. Black-Right-Pointing-Pointer Oct4 and Nanog are up-regulated via de novo synthesis by cell cycle interruption.« less

Microarray analysis on gene regulation by estrogen, progesterone and tamoxifen in human endometrial stromal cells.

PubMed

Ren, Chun-E; Zhu, Xueqiong; Li, Jinping; Lyle, Christian; Dowdy, Sean; Podratz, Karl C; Byck, David; Chen, Hai-Bin; Jiang, Shi-Wen

2015-03-13

Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

Space environment effect on cell cycle of proliferating FRTL-5 cells

NASA Astrophysics Data System (ADS)

Curcio, Francesco; Saverio Ambesi-Impiombato, Francesco; Meli, Antonella; Perrella, Giuseppina; Spelat, Renza; Zambito, Anna Maria

The space environment is a unique laboratory to study the response of living organisms to microgravity and cosmic radiation at the molecular and cellular levels. Significant results obtained by us during the Eneide Mission (Soyuz 9S and 10S 2005) showed a different sensitivity to space environment of cells in proliferative state as compared to those in physiological stand-by. The main object of our investigation was to validate these important findings and to study the molecular mechanisms underlying the phenomenon. To this purpose, a cell model of normal cells derived from rat thyroids which can be kept unattended for up to 20 days in a proliferative medium and at room temperature (FRTL-5) were used in a 10 days experiment on a FOTON satellite and in a 15 days experiment in the STS-120 shuttle mission. Experimental design for both flights was planned on the basis of the "ENEIDE" mission results. Microarray analysis has been performed on the samples from Foton M3 and STS-120. Background subtraction, quality assessment and normalization as well as the definition of specific evaluation algorithms have been performed. Based on the hyper G Test function we computed the Hyper geometric p-values for over representation of genes at all Gene Ontology (GO) terms in the induced GO graphs; this test was performed for each GO category and applied also to KEGG pathways. Results show the good quality of the experiment and our data show that the pathways mostly affected by the flight are: a) the cell cycle, b) the ubiquitin mediated proteolysis, c) the repair mechanisms, d) the adherens junction and e) the pyrimidine metabolism. The patways studied indicate that the cells suffer a slowing of cell cycle as well as upregulation of the DNA and RNA repair processes and even further corroborate the validity of using the FRTL5 cells as biosensors for monitoring the effectiveness of countermeasures to damage caused by the Space.

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed Central

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-01-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA. PMID:8943327

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

PubMed

Pasion, S G; Hines, J C; Ou, X; Mahmood, R; Ray, D S

1996-12-01

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

[Effect of different oxygen concentrations on biological properties of bone marrow hematopoietic stem cells of mice].

PubMed

Ma, Yi-Ran; Ren, Si-Hua; He, Yu-Xin; Wang, Lin-Lin; Jin, Li; Hao, Yi-Wen

2012-10-01

This study purposed to investigate the effects of different oxygen concentrations and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and their possible mechanisms through simulating oxygen environment to which the peripheral blood HSC are subjected in peripheral blood HSCT. The proliferation ability, cell cycle, directed differentiation ability, ROS level and hematopoietic reconstitution ability of Lin(-)c-kit(+)Sca-1(+) BMHSC were detected by using in vitro amplification test, directional differentiation test, cell cycle analysis, ROS assay and transplantation of Lin(-)c-kit(+)Sca-1(+) HSC from sublethally irradiated mice respectively. The results showed that oxygen concentrations lower than normal oxygen concentration, especially in hypoxic oxygen environment, could reduce ROS generation and amplify more primitive CD34(+)AC133(+) HSC and active CD34(+) HSC, and maintain more stem cells in the G(0)/G(1) phase, which is more helpful to the growth of CFU-S and viability of mice. At the same time, BMHSC exposed to normal oxygen level or inconstant and greatly changed oxygen concentrations could produce a high level of ROS, and the above-mentioned features and functional indicators are relatively low. It is concluded that ROS levels of HSC in BMHSCT are closely related with the oxygen concentration surrounding the cells and its stability. Low oxygen concentration and antioxidant intervention are helpful to transplantation of BMHSC.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

PubMed

Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

2018-05-07

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells.

PubMed

Shimura, Tsutomu; Hamada, Nobuyuki; Sasatani, Megumi; Kamiya, Kenji; Kunugita, Naoki

2014-01-01

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G 1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G 1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

PubMed Central

Shimura, Tsutomu; Hamada, Nobuyuki; Sasatani, Megumi; Kamiya, Kenji; Kunugita, Naoki

2014-01-01

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation. PMID:24583467

The long non-coding RNA HOTAIR promotes the proliferation of serous ovarian cancer cells through the regulation of cell cycle arrest and apoptosis

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

Qiu, Jun-jun; Department of Obstetrics and Gynecology of Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, 413 Zhaozhou Road, Shanghai 200011

HOX transcript antisense RNA (HOTAIR) is a well-known long non-coding RNA (lncRNA) whose dysregulation correlates with poor prognosis and malignant progression in many forms of cancer. Here, we investigate the expression pattern, clinical significance, and biological function of HOTAIR in serous ovarian cancer (SOC). Clinically, we found that HOTAIR levels were overexpressed in SOC tissues compared with normal controls and that HOTAIR overexpression was correlated with an advanced FIGO stage and a high histological grade. Multivariate analysis revealed that HOTAIR is an independent prognostic factor for predicting overall survival in SOC patients. We demonstrated that HOTAIR silencing inhibited A2780 andmore » OVCA429 SOC cell proliferation in vitro and that the anti-proliferative effects of HOTAIR silencing also occurred in vivo. Further investigation into the mechanisms responsible for the growth inhibitory effects by HOTAIR silencing revealed that its knockdown resulted in the induction of cell cycle arrest and apoptosis through certain cell cycle-related and apoptosis-related proteins. Together, these results highlight a critical role of HOTAIR in SOC cell proliferation and contribute to a better understanding of the importance of dysregulated lncRNAs in SOC progression. - Highlights: • HOTAIR overexpression correlates with an aggressive tumour phenotype and a poor prognosis in SOC. • HOTAIR promotes SOC cell proliferation both in vitro and in vivo. • The proliferative role of HOTAIR is associated with regulation of the cell cycle and apoptosis.« less

Transcriptional analysis of the conidiation pattern shift of the entomopathogenic fungus Metarhizium acridum in response to different nutrients.

PubMed

Wang, Zhenglong; Jin, Kai; Xia, Yuxian

2016-08-09

Most fungi, including entomopathogenic fungi, have two different conidiation patterns, normal and microcycle conidiation, under different culture conditions, eg, in media containing different nutrients. However, the mechanisms underlying the conidiation pattern shift are poorly understood. In this study, Metarhizium acridum undergoing microcycle conidiation on sucrose yeast extract agar (SYA) medium shifted to normal conidiation when the medium was supplemented with sucrose, nitrate, or phosphate. By linking changes in nutrients with the conidiation pattern shift and transcriptional changes, we obtained conidiation pattern shift libraries by Solexa/Illumina deep-sequencing technology. A comparative analysis demonstrated that the expression of 137 genes was up-regulated during the shift to normal conidiation, while the expression of 436 genes was up-regulated at the microcycle conidiation stage. A comparison of subtractive libraries revealed that 83, 216, and 168 genes were related to sucrose-induced, nitrate-induced, and phosphate-induced conidiation pattern shifts, respectively. The expression of 217 genes whose expression was specific to microcycle conidiation was further analyzed by the gene expression profiling via multigene concatemers method using mRNA isolated from M. acridum grown on SYA and the four normal conidiation media. The expression of 142 genes was confirmed to be up-regulated on standard SYA medium. Of these 142 genes, 101 encode hypothetical proteins or proteins of unknown function, and only 41 genes encode proteins with putative functions. Of these 41 genes, 18 are related to cell growth, 10 are related to cell proliferation, three are related to the cell cycle, three are related to cell differentiation, two are related to cell wall synthesis, two are related to cell division, and seven have other functions. These results indicate that the conidiation pattern shift in M. acridum mainly results from changes in cell growth and proliferation. The results indicate that M. acridum shifts conidiation pattern from microcycle conidiation to normal conidiation when there is increased sucrose, nitrate, or phosphate in the medium during microcycle conidiation. The regulation of conidiation patterning is a complex process involving the cell cycle and metabolism of M. acridum. This study provides essential information about the molecular mechanism of the induction of the conidiation pattern shift by single nutrients.

Differential effects of selenite and selenate on human melanocytes, keratinocytes, and melanoma cells.

PubMed

Bandura, Laura; Drukala, Justyna; Wolnicka-Glubisz, Agnieszka; Björnstedt, Mikael; Korohoda, Wlodzimierz

2005-04-01

Among the substances that attracted the attention of oncologists in recent years are selenium-containing compounds, both inorganic and organic. Several epidemiological studies have shown an inverse correlation between selenium intake and cancer incidence. In the experiments reported here, we compared the effects of 2 inorganic selenium-containing salts that differed in the level of selenium oxidation, selenite IV and selenate VI. We tested the effects of these 2 compounds on cell survival and growth, cell cycle processing, cell morphology, cytoskeleton, and lipid peroxidation in 3 human skin cell types: normal keratinocytes, melanocytes, and human melanoma cell line HTB140. The different effects of selenite and selenate on the viability, growth, and morphology of normal cells and tumor cells are reported and provide a base for future research and treatment of some neoplastic diseases. The attention is paid to cell apoptosis induced by selenite and not by selenate, and the effects of tested substances on thioredoxin reductase system are postulated.

The role of methionine metabolism in inflammatory bowel disease

USDA-ARS?s Scientific Manuscript database

Methionine (Met) cycle activity is critical for normal cell functions. Met metabolites S-adenosylmethionine (SAM) and methylthioadenosine (MTA) are anti-inflammatory, yet their role in inflammatory bowel disease (IBD) is poorly understood. We hypothesize that active IBD leads to changes in Met metab...

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.

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells.

PubMed

Morris, Brett A; Burkel, Brian; Ponik, Suzanne M; Fan, Jing; Condeelis, John S; Aguirre-Ghiso, Julio A; Castracane, James; Denu, John M; Keely, Patricia J

2016-11-01

Increased breast density attributed to collagen I deposition is associated with a 4-6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Regulatory Response to Carbon Starvation in Caulobacter crescentus

PubMed Central

Britos, Leticia; Abeliuk, Eduardo; Taverner, Thomas; Lipton, Mary; McAdams, Harley; Shapiro, Lucy

2011-01-01

Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells. PMID:21494595

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

PubMed

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

2006-05-01

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

The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

PubMed Central

Gorgescu, Walter; Tang, Jonathan; Costes, Sylvain V.; Karpen, Gary H.

2012-01-01

CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote. PMID:23300382

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

PubMed Central

Adeyemi, Richard O.; Pintel, David J.

2014-01-01

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication. PMID:24415942

Parvovirus-induced depletion of cyclin B1 prevents mitotic entry of infected cells.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-01-01

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.

Core exosome-independent roles for Rrp6 in cell cycle progression.

PubMed

Graham, Amy C; Kiss, Daniel L; Andrulis, Erik D

2009-04-01

Exosome complexes are 3' to 5' exoribonucleases composed of subunits that are critical for numerous distinct RNA metabolic (ribonucleometabolic) pathways. Several studies have implicated the exosome subunits Rrp6 and Dis3 in chromosome segregation and cell division but the functional relevance of these findings remains unclear. Here, we report that, in Drosophila melanogaster S2 tissue culture cells, dRrp6 is required for cell proliferation and error-free mitosis, but the core exosome subunit Rrp40 is not. Micorarray analysis of dRrp6-depleted cell reveals increased levels of cell cycle- and mitosis-related transcripts. Depletion of dRrp6 elicits a decrease in the frequency of mitotic cells and in the mitotic marker phospho-histone H3 (pH3), with a concomitant increase in defects in chromosome congression, separation, and segregation. Endogenous dRrp6 dynamically redistributes during mitosis, accumulating predominantly but not exclusively on the condensed chromosomes. In contrast, core subunits localize predominantly to MTs throughout cell division. Finally, dRrp6-depleted cells treated with microtubule poisons exhibit normal kinetochore recruitment of the spindle assembly checkpoint protein BubR1 without restoring pH3 levels, suggesting that these cells undergo premature chromosome condensation. Collectively, these data support the idea that dRrp6 has a core exosome-independent role in cell cycle and mitotic progression.

DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

PubMed Central

Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

2013-01-01

Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment. PMID:23675572

FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential

PubMed Central

Lay, Kenneth; Kume, Tsutomu; Fuchs, Elaine

2016-01-01

Adult tissue stem cells (SCs) reside in niches, which orchestrate SC behavior. SCs are typically used sparingly and exist in quiescence unless activated for tissue growth. Whether parsimonious SC use is essential to conserve long-term tissue-regenerating potential during normal homeostasis remains poorly understood. Here, we examine this issue by conditionally ablating a key transcription factor Forkhead box C1 (FOXC1) expressed in hair follicle SCs (HFSCs). FOXC1-deficient HFSCs spend less time in quiescence, leading to markedly shortened resting periods between hair cycles. The enhanced hair cycling accelerates HFSC expenditure, and impacts hair regeneration in aging mice. Interestingly, although FOXC1-deficient HFs can still form a new bulge that houses HFSCs for the next hair cycle, the older bulge is left unanchored. As the new hair emerges, the entire old bulge, including its reserve HFSCs and SC-inhibitory inner cell layer, is lost. We trace this mechanism first, to a marked increase in cell cycle-associated transcripts upon Foxc1 ablation, and second, to a downstream reduction in E-cadherin–mediated inter-SC adhesion. Finally, we show that when the old bulge is lost with each hair cycle, overall levels of SC-inhibitory factors are reduced, further lowering the threshold for HFSC activity. Taken together, our findings suggest that HFSCs have restricted potential in vivo, which they conserve by coupling quiescence to adhesion-mediated niche maintenance, thereby achieving long-term tissue homeostasis. PMID:26912458

Anti-tumor effects of osthole on ovarian cancer cells in vitro.

PubMed

Jiang, Guoqiang; Liu, Jia; Ren, Baoyin; Tang, Yawei; Owusu, Lawrence; Li, Man; Zhang, Jing; Liu, Likun; Li, Weiling

2016-12-04

Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat gynecological disease in some countries. Osthole, an active O-methylated coumadin isolated from Cnidium monnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-seizure and anti-inflammatory effects. However, the anti-tumor mechanism of osthole is not well known. Here, we show that osthole inhibited the proliferation and migration of two widely used ovarian cancer cell lines, A2780 and OV2008 cells, in a dose-dependent manner. The study investigated the molecular mechanisms underlying ovarian cancer cells proliferation, apoptosis, cell cycle arrest and migration triggered by osthole. Ovarian cancer cell lines A2780, OV2008 and normal ovarian cell line IOSE80 were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry assays were performed to confirm apoptosis and cell cycle. We employed wound healing and transwell assays to delineate invasive and migratory potential triggered by osthole. MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with osthole without effect on normal ovarian cells. Flow cytometric analysis revealed that osthole suppressed cells proliferation by promoting G2/M arrest and inducing apoptosis. The underlying mechanisms involved were regulation of the relative apoptotic protein Bcl-2, Bax and Caspase 3/9. In addition, wound healing and transwell assays revealed that the migratory potential and activity of matrix metalloproteinase MMP-2 and MMP-9 were markedly inhibited when cells were exposed to osthole. Our findings suggested that osthole has the potential to be used in novel anti-cancer therapeutic formulations for ovarian cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat.

PubMed

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan; Li, P Andy; Sun, Tao

2013-11-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome.

Ornithine decarboxylase activity in rat organs and tissues under artificial hypobiosis.

PubMed

Aksyonova, G E; Logvinovich, O S; Fialkovskaya, L A; Afanasyev, V N; Ignat'ev, D A; Kolomiytseva, I K

2010-09-01

The influence of hypothermia-hypoxia-hypercapnia on ornithine decarboxylase (ODC, EC 4.1.1.17) activities in rat organs and tissues and also on the thymocyte distribution throughout the cell cycle stages was studied. The state of artificial hypobiosis in rats on decrease in the body temperature to 14.4-18.0°C during 3.0-3.5 h was accompanied by drops in the ODC activities in the neocortex and liver by 50-60% and in rapidly proliferating tissues (thymus, spleen, and small intestine mucosa) by 80% of the control value. In kidneys the ODC activity raised to 200% of the control level. Twenty-four hours after termination of the cooling and replacing the rats under the standard conditions, the ODC activities in the neocortex, liver, kidneys, spleen, and intestinal mucosa returned to the control values, but remained decreased in the thymus. Forty-eight hours later the ODC activities in the thymus and spleen exceeded the normal level. The distribution of thymocytes throughout the cell cycle stages did not change in rats in the state of hypothermia (hypobiosis); 24 and 48 h after termination of the cooling the fraction of thymocytes in the S stage was decreased and the fraction of the cells in the G(0)+G(1) stage was increased. The normal distribution of thymocytes throughout the cell cycle stages recovered in 72 h. Thus, in the thymus the diminution of the ODC activity preceded the suppression of the cell proliferation rate. The tissue-specific changes in the ODC activity are suggested to reflect adaptive changes in the functional and proliferative activities of organs and tissues during the development of hypobiosis under conditions of hypothermia-hypoxia-hypercapnia.

Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

PubMed

Ozbun, Michelle A; Patterson, Nicole A

2014-08-01

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley & Sons, Inc.

Generation of hair cells in neonatal mice by β-catenin overexpression in Lgr5-positive cochlear progenitors

PubMed Central

Shi, Fuxin; Hu, Lingxiang; Edge, Albert S. B.

2013-01-01

Mammalian hair cells do not regenerate, and their loss is a major cause of deafness. We recently identified leucine-rich repeat containing, G-protein-coupled receptor 5 (Lgr5)-expressing cochlear supporting cells with the capacity for self-renewal and hair cell differentiation in vitro. We found that these cells, a subset of cochlear supporting cells, were responsive to Wnt signaling. Here we asked whether these Lgr5-positive cells, despite their lack of contribution to hair cell replacement after degenerative loss, could be driven by forced expression of β-catenin to act as hair cell progenitors in vivo. We showed that forced stabilization of β-catenin in supporting cells in neonatal animals resulted in proliferation of supporting cells and generation of hair cells. Although β-catenin expression was increased by genetic means in all supporting cells, entry to the cell cycle and differentiation to hair cells of the normally postmitotic cells was restricted to the Lgr5-positive population. Our finding suggests that Wnt/β-catenin can drive Lgr5-positive cells to act as hair cell progenitors, even after their exit from the cell cycle and apparent establishment of cell fate. PMID:23918377

The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication.

PubMed

Bercik, P; Park, A J; Sinclair, D; Khoshdel, A; Lu, J; Huang, X; Deng, Y; Blennerhassett, P A; Fahnestock, M; Moine, D; Berger, B; Huizinga, J D; Kunze, W; McLean, P G; Bergonzelli, G E; Collins, S M; Verdu, E F

2011-12-01

The probiotic Bifidobacterium longum NCC3001 normalizes anxiety-like behavior and hippocampal brain derived neurotrophic factor (BDNF) in mice with infectious colitis. Using a model of chemical colitis we test whether the anxiolytic effect of B. longum involves vagal integrity, and changes in neural cell function. Methods  Mice received dextran sodium sulfate (DSS, 3%) in drinking water during three 1-week cycles. Bifidobacterium longum or placebo were gavaged daily during the last cycle. Some mice underwent subdiaphragmatic vagotomy. Behavior was assessed by step-down test, inflammation by myeloperoxidase (MPO) activity and histology. BDNF mRNA was measured in neuroblastoma SH-SY5Y cells after incubation with sera from B. longum- or placebo-treated mice. The effect of B. longum on myenteric neuron excitability was measured using intracellular microelectrodes. Chronic colitis was associated with anxiety-like behavior, which was absent in previously vagotomized mice. B. longum normalized behavior but had no effect on MPO activity or histological scores. Its anxiolytic effect was absent in mice with established anxiety that were vagotomized before the third DSS cycle. B. longum metabolites did not affect BDNF mRNA expression in SH-SY5Y cells but decreased excitability of enteric neurons. In this colitis model, anxiety-like behavior is vagally mediated. The anxiolytic effect of B. longum requires vagal integrity but does not involve gut immuno-modulation or production of BDNF by neuronal cells. As B. longum decreases excitability of enteric neurons, it may signal to the central nervous system by activating vagal pathways at the level of the enteric nervous system. © 2011 Blackwell Publishing Ltd.

Role of HLA-G1 in trophoblast cell proliferation, adhesion and invasion

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

Jiang, Feng, E-mail: jiangfeng1161@163.com; Zhao, Hongxi; Wang, Li

Trophoblast cells are important in embryo implantation and fetomaternal tolerance. HLA-G is specifically expressed at the maternal–fetal interface and is a regulator in pregnancy. The aim of the present study was to detect the effect of HLA-G1 on trophoblast cell proliferation, adhesion, and invasion. Human trophoblast cell lines (JAR and HTR-8/SVneo cells) were infected with HLA-G1-expressing lentivirus. After infection, HLA-G1 expression of the cells was detected by western blotting. Cell proliferation was detected by the BrdU assay. The cell cycle and apoptosis of JAR and HTR-8/SVneo cells was measured by flow cytometry (FCM). The invasion of the cells under different conditionsmore » was detected by the transwell invasion chamber assay. HLA-G1 didn't show any significant influence on the proliferation, apoptosis, adhesion, and invasion of trophocytes in normal culture conditions. However, HLA-G1 inhibited JAR and HTR-8/SVneo cells invasion induced by hepatocyte growth factor (HGF) under normal oxygen conditions. In conditions of hypoxia, HLA-G1 couldn't inhibit the induction of cell invasion by HGF. HLA-G1 is not an independent factor for regulating the trophocytes. It may play an indirect role in embryo implantation and formation of the placenta. - Highlights: • HLA-G1 could not influence trophocytes under normal conditions. • HLA-G1 inhibited cell invasion induced by HGF under normal oxygen condition. • HLA-G1 could not influence cell invasion under hypoxia conditions.« less

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes.

PubMed

Malini, Maressa; Souza, Marilesia Ferreira de; Oliveira, Marcelo Tempesta de; Antunes, Lusânia Maria Greggi; Figueiredo, Suely Gomes de; Barbosa, Aneli M; Dekker, Robert F H; Cólus, Ilce Mara de Syllos

2015-01-01

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

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

NOK mediates glycolysis and nuclear PDC associated histone acetylation.

PubMed

Shi, Wei-Ye; Yang, Xiao; Huang, Bo; Shen, Wen H; Liu, Li

2017-06-01

NOK is a potent oncogene that can transform normal cells to cancer cells. We hypothesized that NOK might impact cancer cell metabolism and histone acetylation. We show that NOK localizes in the mitochondria, and while it minimally impacts tricarboxylic acid (TCA) cycle, it markedly inhibits the process of electron transport and oxidative phosphorylation processes and dramatically enhances aerobic glycolysis in cancer cells. NOK promotes the mitochondrial-nuclear translocation of pyruvate dehydrogenase complex (PDC), and enhances histone acetylation in the nucleus. Together, these findings show that NOK mediates glycolysis and nuclear PDC associated histone acetylation.

Inefficient differentiation response to cell cycle stress leads to genomic instability and malignant progression of squamous carcinoma cells

PubMed Central

Alonso-Lecue, Pilar; de Pedro, Isabel; Coulon, Vincent; Molinuevo, Rut; Lorz, Corina; Segrelles, Carmen; Ceballos, Laura; López-Aventín, Daniel; García-Valtuille, Ana; Bernal, José M; Mazorra, Francisco; Pujol, Ramón M; Paramio, Jesús; Ramón Sanz, J; Freije, Ana; Toll, Agustí; Gandarillas, Alberto

2017-01-01

Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy. PMID:28661481

Palladium Nanoparticles Induce Disturbances in Cell Cycle Entry and Progression of Peripheral Blood Mononuclear Cells: Paramount Role of Ions

PubMed Central

Clemente, Emanuela; Di Giampaolo, Luca; Mariani-Costantini, Renato; Leopold, Kerstin; Schindl, Roland; Lotti, Lavinia V.; Sabbioni, Enrico; Niu, Qiao; Di Gioacchino, Mario

2014-01-01

There is concern about the possible toxicity of palladium nanoparticles (Pd-NP), as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV) ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV) exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV) ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV) ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations. PMID:25105151

RECQL4-deficient cells are hypersensitive to oxidative stress/damage: Insights for osteosarcoma prevalence and heterogeneity in Rothmund-Thomson syndrome

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

Werner, Sean R.; Prahalad, Agasanur K.; Yang Jieping

2006-06-23

Rothmund-Thomson syndrome (RTS) is a heterogeneous disease, associated with increased prevalence of osteosarcoma in very young patients with a mutated RECQL4 gene. In this study, we tested the ability of RECQL4 deficient fibroblasts, derived from a RTS patient to recover from hydrogen peroxide (H{sub 2}O{sub 2})-induced oxidative stress/damage. Immunoperoxidase staining for 8-oxo-deoxyguanosine (8-oxo-dG) formation in RTS and normal human fibroblasts were compared to assess DNA damage. We determined DNA synthesis, cell growth, cell cycle distribution, and viability in RTS and normal human fibroblasts before and after H{sub 2}O{sub 2} treatment. H{sub 2}O{sub 2} induces 8-oxo-dG formation in both RTS andmore » normal fibroblasts. In normal human fibroblasts, RECQL4 was predominantly localized to cytoplasm; nuclear translocation and foci formation occurred in response to oxidant stimulation. After recovery from oxidant exposure, viable RTS fibroblasts showed irreversible growth arrest compared to normal fibroblasts. DNA synthesis decreased significantly in treated RTS cells, with concomitant reduction of cells in the S-phase. These results suggest that enhanced oxidant sensitivity in RECQL4 deficient fibroblasts derived from RTS patients could be attributed to abnormal DNA metabolism and proliferation failure. The ramifications of these findings on osteosarcoma prevalence and heterogeneity in RTS are discussed.« less

E2F Activators Signal and Maintain Centrosome Amplification in Breast Cancer Cells

PubMed Central

Lee, Mi-Young; Moreno, Carlos S.

2014-01-01

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2+ cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2+ cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2+ breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2. PMID:24797070

The combined effects of single-nucleotide polymorphisms, tobacco products, and ethanol on normal resting blood mononuclear cells.

PubMed

Cederblad, Lena; Thunberg, Ulf; Engström, Mats; Castro, Juan; Rutqvist, Lars Erik; Laytragoon-Lewin, Nongnit

2013-05-01

Tobacco and ethanol consumption are crucial factors in the development of various diseases including cancer. In this investigation, we evaluated the combined effects of a number of single nucleotide polymorphisms (SNPs), with ethanol and tobacco products on healthy individuals. Pure nicotine, cigarette smoke extract, and Swedish snuff (snus) extract were used. The effects were examined by means of in vitro cell cycle progression and cell death of peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. After 3 days, in vitro, resting PBMCs entered the S and G2 stage in the presence of 100 µM nicotine. The PBMCs only proceeded to S stage, in the presence of 0.2% ethanol. The nicotine- and ethanol-induced normal cell cycle progression correlated to a number of SNPs in the IL12RB2, Rad 52, XRCC2, P53, CCND3, and ABCA1 genes. Certain SNPs in Caspases 8, IL12RB2, Rad 52, MMP2, and MDM2 genes appeared to significantly influence the effects of EtOH-, snus-, and snus + EtOH-induced cell death. Importantly, the highest degree of cell death was observed in the presence of smoke + EtOH. The amount of cell death under this treatment condition also correlated to specific SNPs, located in the MDM2, ABCA1, or GASC1 genes. Cigarette smoke in combination with ethanol strongly induced massive cell death. Long-term exposure to smoke and ethanol could provoke chronic inflammation, and this could be the initiation of disease including the development of cancer at various sites.

Anti-mitotic agents: Are they emerging molecules for cancer treatment?

PubMed

Penna, Larissa Siqueira; Henriques, João Antonio Pêgas; Bonatto, Diego

2017-05-01

Mutations in cancer cells frequently result in cell cycle alterations that lead to unrestricted growth compared to normal cells. Considering this phenomenon, many drugs have been developed to inhibit different cell-cycle phases. Mitotic phase targeting disturbs mitosis in tumor cells, triggers the spindle assembly checkpoint and frequently results in cell death. The first anti-mitotics to enter clinical trials aimed to target tubulin. Although these drugs improved the treatment of certain cancers, and many anti-microtubule compounds are already approved for clinical use, severe adverse events such as neuropathies were observed. Since then, efforts have been focused on the development of drugs that also target kinases, motor proteins and multi-protein complexes involved in mitosis. In this review, we summarize the major proteins involved in the mitotic phase that can also be targeted for cancer treatment. Finally, we address the activity of anti-mitotic drugs tested in clinical trials in recent years. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of propionic acid and valeric acid on the cell cycle in root meristems of Pisum sativum

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

Tramontano, W.A.; Yang, Shauyu; Delillo, A.R.

1990-01-01

Propionic acid and valeric acid at 1mM reduced the mitotic index of root meristem cells of Pisum sativum to < 1% after 12 hr in aerated White's medium. This effect varied with different acid concentrations. After a 12 hr exposure to either acid, seedlings transferred to fresh medium without either acid, resumed their normal mitotic index after 12 hr, with a burst of mitosis 8 hr post-transfer. Exposure of root meristem cells to either acid also inhibited ({sup 3}H)-TdR incorporation. Neither acid significantly altered the distribution of meristematic cells in G1 and G2 after 12 hr. The incorporation of ({supmore » 3}H) - uridine was also unaltered by the addition of either acid. This information suggests that propionic acid and valeric acid, limit progression through the cell cycle by inhibiting DNA synthesis and arresting cells in G1 and G2. These results were consistent with previous data which utilized butyric acid.« less

[Radiation-induced genomic instability: phenomenon, molecular mechanisms, pathogenetic significance].

PubMed

Mazurik, V K; Mikhaĭlov, V F

2001-01-01

The recent data on the radiation-induced genome instability as a special state of progeny of cells irradiated in vitro as well as after a whole body exposure to ionizing radiation, that make these cells considerably different from normal, unirradiated cells, were considered. This state presents a number of cytogenetical, molecular-biological, cytological and biochemical manifestations untypical for normal cells. The state is controlled by the mechanisms of regulation of checkpoints of cell cycle, and apoptosis, that is under gene p53 control. The proof has been found that this state transfers from irradiated maternal cells to their surviving progeny by the epigenetical mechanisms and would exist until the cells restore the original state of response on the DNA damage. From the point of view of the genome instability conception, that considers the chromatine rearrangement as the adaptive-evolution mechanism of adaptation of the species to changeable environmental conditions, the radiation-induced genome instability may be considered as transition of irradiated progeny to the state of read these to adaptation changes with two alternative pathways. The first leads to adaptation to enviromental conditions and restoring of normal cell functions. The second presents the cell transition into the transformed state with remain genome instability and with increase of tumour growth probability.

Improving cycle stability of SnS anode for sodium-ion batteries by limiting Sn agglomeration

NASA Astrophysics Data System (ADS)

Wang, Wenhui; Shi, Liang; Lan, Danni; Li, Quan

2018-02-01

Flower-like SnS nanostructures are obtained by a simple solvothermal method for anode applications in Na-ion batteries. We show experimental evidence of progressive Sn agglomeration and crystalline Na2S enrichment at the end of de-sodiation process of the SnS electrode, both of which contribute to the capacity decay of the electrode upon repeated cycles. By replacing the commonly adopted acetylene black conductive additive with multi-wall carbon nanotubes (MWCNT), the cycle stability of the SnS electrode is largely improved, which correlates well with the observed suppression of both Sn agglomeration and Na2S enrichment at the end of de-sodiation cycle. A full cell is assembled with the SnS/MWCNT anode and the P2-Na2/3Ni1/3Mn1/2Ti1/6O2 cathode. An initial energy density of 262 Wh/kg (normalized to the total mass of cathode and anode) is demonstrated for the full cell, which retains 71% of the first discharge capacity after 40 cycles.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

PubMed

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2014-06-30

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

SCFFBXW7α modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability

PubMed Central

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

2014-01-01

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint. PMID:24970797

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

PubMed Central

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

PubMed

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of Schizosaccharomyces pombe Meiosis.

PubMed

Yamashita, Akira; Sakuno, Takeshi; Watanabe, Yoshinori; Yamamoto, Masayuki

2017-09-01

Meiosis is a specialized cell cycle that generates haploid gametes from diploid cells. The fission yeast Schizosaccharomyces pombe is one of the best model organisms for studying the regulatory mechanisms of meiosis. S. pombe cells, which normally grow in the haploid state, diploidize by conjugation and initiate meiosis when starved for nutrients, especially nitrogen. Following two rounds of chromosome segregation, spore formation takes place. The switch from mitosis to meiosis is controlled by a kinase, Pat1, and an RNA-binding protein, Mei2. Mei2 is also a key factor for meiosis-specific gene expression. Studies on S. pombe have offered insights into cell cycle regulation and chromosome segregation during meiosis. Here we outline the current understanding of the molecular mechanisms regulating the initiation and progression of meiosis, and introduce methods for the study of meiosis in fission yeast. © 2017 Cold Spring Harbor Laboratory Press.

A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts

PubMed Central

Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna; Schrøder, Henrik Daa

2013-01-01

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts. PMID:23717533

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

PubMed Central

Malliaras, Konstantinos; Zhang, Yiqiang; Seinfeld, Jeffrey; Galang, Giselle; Tseliou, Eleni; Cheng, Ke; Sun, Baiming; Aminzadeh, Mohammad; Marbán, Eduardo

2013-01-01

Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation. PMID:23255322

Life cycle test results of a bipolar nickel hydrogen battery

NASA Technical Reports Server (NTRS)

Cataldo, R. L.

1985-01-01

A history is given of low Earth orbit (LEO) laboratory test data on a 6.5 ampere-hour bipolar nickel hydrogen battery designed and built at the NASA Lewis Research Center. The bipolar concept is a means of achieving the goal of producing an acceptable battery, of higher energy density, able to withstand the demands of low-Earth-orbit regimes. Over 4100 LEO cycles were established on a ten cell battery. It seems that any perturbation on normal cycling effects the cells performance. Explanations and theories of the battery's behavior are varied and widespread among those closely associated with it. Deep discharging does provide a reconditioning effect and further experimentation is planned in this area. The battery watt-hour efficiency is about 75 percent and the time averaged, discharge voltage is about 1.26 volts for all cells at both the C/4 and LEO rate. Since a significant portion of the electrode capacity has degraded, the LEO cycle discharges are approaching depths of 90 to 100 percent of the high rate capacity. Therefore, the low end-of-discharge voltages occur precipitously after the knee of the discharge curve and is more an indication of electrode capacity and is a lesser indicator of overall cell performance.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

PubMed

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

2013-10-22

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

Preclinical evaluation of transcriptional targeting strategy for human hepatocellular carcinoma in an orthotopic xenograft mouse model.

PubMed

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

2013-08-01

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

Estrous Cyclicity of Mice During Simulated Weightlessness

NASA Technical Reports Server (NTRS)

Moyer, Eric; Talyansky, Yuli; Scott, Ryan; Tash, Joseph; Christenson, Lane; Alwood, Joshua; Ronca, April

2017-01-01

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n22), normally loaded HU Cage Pair-Fed controls (CPF; n22), and Vivarium controls fed ad libitum (VIV; n10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, andor due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5 vs 3.0, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system.

BOD1 Is Required for Cognitive Function in Humans and Drosophila

PubMed Central

Motazacker, M. Mahdi; Nijhof, Bonnie; Castells-Nobau, Anna; Asztalos, Zoltan; Weißmann, Robert; Behjati, Farkhondeh; Tzschach, Andreas; Felbor, Ute; Scherthan, Harry; Sayfati, Seyed Morteza; Ropers, H. Hilger.; Kahrizi, Kimia; Najmabadi, Hossein; Swedlow, Jason R.; Schenck, Annette; Kuss, Andreas W.

2016-01-01

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features. PMID:27166630

The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

PubMed

Chou, Chu-Fang; Hsieh, Yu-Hua; Grubbs, Clinton J; Atigadda, Venkatram R; Mobley, James A; Dummer, Reinhard; Muccio, Donald D; Eto, Isao; Elmets, Craig A; Garvey, W Timothy; Chang, Pi-Ling

2018-06-01

Bexarotene (Targretin ® ) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC 50 ) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients.

PubMed

Tu, Chengjian; Mojica, Wilfrido; Straubinger, Robert M; Li, Jun; Shen, Shichen; Qu, Miao; Nie, Lei; Roberts, Rick; An, Bo; Qu, Jun

2017-05-01

The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

miR-181b Promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients

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

Wang, Baocan; Li, Wenxi; Guo, Kun

2012-04-27

Highlights: Black-Right-Pointing-Pointer miR-181a and miR-181b, especially, miR-181b could be induced by transforming growth factor-beta 1 (TGF-{beta}1) in hepatic stellate cells. Black-Right-Pointing-Pointer miR-181b could promote HSC-T6 cell proliferation by directly targeting the negative cell regulator-p27 in HSC-T6 cell. Black-Right-Pointing-Pointer miR-181b was identified as potential serum diagnostic marker for liver cirrhosis patients. -- Abstract: MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because ofmore » the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-{beta}1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-{beta}1 pathway by a currently unknown mechanism.« less

Jaceosidin, isolated from dietary mugwort (Artemisia princeps), induces G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation.

PubMed

Lee, Jong-Gyu; Kim, Ji-Hyun; Ahn, Ji-Hye; Lee, Kyung-Tae; Baek, Nam-In; Choi, Jung-Hye

2013-05-01

Jaceosidin, a flavonoid derived from Artemisia princeps (Japanese mugwort), has been shown to inhibit the growth of several human cancer cells, However, the exact mechanism for the cytotoxic effect of jaceosidin is not completely understood. In this study, we investigated the molecular mechanism involved in the antiproliferative effect of jaceosidin in human endometrial cancer cells. We demonstrated that jaceosidin is a more potent inhibitor of cell growth than cisplatin in human endometrial cancer cells. In contrast, jaceosidin-induced cytotoxicity in normal endometrial cells was lower than that observed for cisplatin. Jaceosidin induced G2/M phase cell cycle arrest and modulated the levels of cyclin B and p-Cdc2 in Hec1A cells. Knockdown of p21 using specific siRNAs partially abrogated jaceosidin-induced cell growth inhibition. Additional mechanistic studies revealed that jaceosidin treatment resulted in an increase in phosphorylation of Cdc25C and ATM-Chk1/2. Ku55933, an ATM inhibitor, reversed jaceosidin-induced cell growth inhibition, in part. Moreover, jaceosidin treatment resulted in phosphorylation of ERK, and pretreatment with the ERK inhibitor, PD98059, attenuated cell growth inhibition by jaceosidin. These data suggest that jaceosidin, isolated from Japanese mugwort, modulates the ERK/ATM/Chk1/2 pathway, leading to inactivation of the Cdc2-cyclin B1 complex, followed by G2/M cell cycle arrest in endometrial cancer cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent Pancreatic Cancer Cells *

PubMed Central

Ewton, Daina Z.; Hu, Jing; Vilenchik, Maria; Deng, Xiaobing; Luk, Kin-chun; Polonskaia, Ann; Hoffman, Ann F.; Zipf, Karen; Boylan, John F.; Friedman, Eileen A.

2011-01-01

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they re-enter the cell cycle. Earlier studies demonstrated that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells, that Mirk uses several mechanisms to block cell cycling, and that Mirk increases expression of antioxidant genes which lower ROS levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1 and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In prior studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. PMID:21878655

Chromosomal Aberrations in Normal and AT Cells Exposed to High Dose of Low Dose Rate Irradiation

NASA Technical Reports Server (NTRS)

Kawata, T.; Shigematsu, N.; Kawaguchi, O.; Liu, C.; Furusawa, Y.; Hirayama, R.; George, K.; Cucinotta, F.

2011-01-01

Ataxia telangiectasia (A-T) is a human autosomally recessive syndrome characterized by cerebellar ataxia, telangiectases, immune dysfunction, and genomic instability, and high rate of cancer incidence. A-T cell lines are abnormally sensitive to agents that induce DNA double strand breaks, including ionizing radiation. The diverse clinical features in individuals affected by A-T and the complex cellular phenotypes are all linked to the functional inactivation of a single gene (AT mutated). It is well known that cells deficient in ATM show increased yields of both simple and complex chromosomal aberrations after high-dose-rate irradiation, but, less is known on how cells respond to low-dose-rate irradiation. It has been shown that AT cells contain a large number of unrejoined breaks after both low-dose-rate irradiation and high-dose-rate irradiation, however sensitivity for chromosomal aberrations at low-dose-rate are less often studied. To study how AT cells respond to low-dose-rate irradiation, we exposed confluent normal and AT fibroblast cells to up to 3 Gy of gamma-irradiation at a dose rate of 0.5 Gy/day and analyzed chromosomal aberrations in G0 using fusion PCC (Premature Chromosomal Condensation) technique. Giemsa staining showed that 1 Gy induces around 0.36 unrejoined fragments per cell in normal cells and around 1.35 fragments in AT cells, whereas 3Gy induces around 0.65 fragments in normal cells and around 3.3 fragments in AT cells. This result indicates that AT cells can rejoin breaks less effectively in G0 phase of the cell cycle? compared to normal cells. We also analyzed chromosomal exchanges in normal and AT cells after exposure to 3 Gy of low-dose-rate rays using a combination of G0 PCC and FISH techniques. Misrejoining was detected in the AT cells only? When cells irradiated with 3 Gy were subcultured and G2 chromosomal aberrations were analyzed using calyculin-A induced PCC technique, the yield of unrejoined breaks decreased in both normal and AT cells and misrejoined breaks increased in both cell lines. The present study suggests that AT cells begin to rejoin breaks when a certain number of breaks are accumulated and an increased number of exchanges were observed in G0 AT cells, which is similar situation after high-dose-rate irradiation.

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

PubMed Central

Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

PubMed Central

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P.; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-01-01

Hypoxia-inducible factor-1α (HIF-1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF-1α remain unclear. β-catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF-1α and β-catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4-2B, were grouped as follows: Negative control (no treatment), HIF-1α overexpression group (transfected with HIF-1α overexpression plasmid) and β-catenin silenced group (transfected with HIF-1α plasmids and β-catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4-2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4-2B cells, transfection with HIF-1α overexpression plasmid led to an enhanced β-catenin nuclear translocation, while β-catenin silencing inhibited β-catenin nuclear translocation. The enhanced β-catenin nuclear translocation induced by HIF-1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non-homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF-1α overexpression enhanced β-catenin nuclear translocation, which led to the activation of the β-catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF-1α overexpression promotes the radioresistance of PCa cells. PMID:29658569

A novel role of KIF3b in the seminoma cell cycle

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

Shen, Hao-Qing; Xiao, Yu-Xi; She, Zhen-Yu

KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed thatmore » the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division. - Highlights: • A significant upregulation of KIF3b is detected in seminoma. • Knockdown of KIF3b impacts on cell proliferation and migration. • KIF3b may have direct impacts upon spindle formation and cytokinesis.« less

Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway.

PubMed

Suriyo, Tawit; Tachachartvanich, Phum; Visitnonthachai, Daranee; Watcharasit, Piyajit; Satayavivad, Jutamaad

2015-12-02

Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells.

PubMed

Zeng, Huawei; Trujillo, Olivia N; Moyer, Mary P; Botnen, James H

2011-01-01

Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 μmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 μmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells. Copyright © 2011, Taylor & Francis Group, LLC

Epidermal cell-shape regulation and subpopulation kinetics during butyrate-induced terminal maturation of normal and SV40-transformed human keratinocytes: epithelial models of differentiation therapy.

PubMed

Staiano-Coico, L; Steinberg, M; Higgins, P J

1990-10-15

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.

D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells

PubMed Central

Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe

2013-01-01

In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent Kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer. PMID:23562856

Effect of nickel chloride on cell proliferation.

PubMed

D'Antò, Vincenzo; Valletta, Rosa; Amato, Massimo; Schweikl, Helmut; Simeone, Michele; Paduano, Sergio; Rengo, Sandro; Spagnuolo, Gianrico

2012-01-01

Metal alloys used in dentistry and in other biomedical fields may release nickel ions in the oral environment. The release of nickel might influence the normal biological and physiological processes, including tissue wound healing, cell growth and proliferation. The aim of this study was to evaluate in vitro the effects of nickel ions on cell cycle, viability and proliferation. Human osteosarcoma cells (U2OS) and human keratinocytes (HaCat) were exposed to different nickel chloride (NiCl(2)) concentrations (0 - 5mM) for various periods exposure. The viability of cultured cells was estimated by flow cytometry using Annexin V-FITC and Propidium Iodide (PI). Cell proliferation was evaluated by using carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometry. Finally, the effects of NiCl(2) on cell cycle were assessed and quantified by flow cytometry. Statistical analysis was performed by means of ANOVA followed by Tukey's test. NiCl(2) induced a dose and time dependent decrease in cell viability. After 24h, 1mM NiCl(2) caused a similar and significant reduction of viability in U2OS and HaCat cells, while higher NiCl(2) concentrations and longer exposure times showed a reduced cytotoxic effect in HaCat as compared to U2OS cells. Exposure to NiCl(2) caused a dose- and time-dependent inhibition of cell proliferation in both cell lines tested, with a prominent effect on U2OS cells. Furthermore, both cell lines exposed to NiCl(2) exhibited significant changes in cell cycle distribution after 24h exposure 2mM NiCl2, as compared to untreated cells (p<0.05). Our results indicate that release of nickel ions may affect cell proliferation. The inhibition of cell growth by NiCl2 is mediated by both cell cycle arrest and by induction of cell death.

Effect of Nickel Chloride on Cell Proliferation

PubMed Central

D’Antò, Vincenzo; Valletta, Rosa; Amato, Massimo; Schweikl, Helmut; Simeone, Michele; Paduano, Sergio; Rengo, Sandro; Spagnuolo, Gianrico

2012-01-01

Objective: Metal alloys used in dentistry and in other biomedical fields may release nickel ions in the oral environment. The release of nickel might influence the normal biological and physiological processes, including tissue wound healing, cell growth and proliferation. The aim of this study was to evaluate in vitro the effects of nickel ions on cell cycle, viability and proliferation. Materials and Methods: Human osteosarcoma cells (U2OS) and human keratinocytes (HaCat) were exposed to different nickel chloride (NiCl2) concentrations (0 - 5mM) for various periods exposure. The viability of cultured cells was estimated by flow cytometry using Annexin V-FITC and Propidium Iodide (PI). Cell proliferation was evaluated by using carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometry. Finally, the effects of NiCl2 on cell cycle were assessed and quantified by flow cytometry. Statistical analysis was performed by means of ANOVA followed by Tukey’s test. Results: NiCl2 induced a dose and time dependent decrease in cell viability. After 24h, 1mM NiCl2 caused a similar and significant reduction of viability in U2OS and HaCat cells, while higher NiCl2 concentrations and longer exposure times showed a reduced cytotoxic effect in HaCat as compared to U2OS cells. Exposure to NiCl2 caused a dose- and time-dependent inhibition of cell proliferation in both cell lines tested, with a prominent effect on U2OS cells. Furthermore, both cell lines exposed to NiCl2 exhibited significant changes in cell cycle distribution after 24h exposure 2mM NiCl2, as compared to untreated cells (p<0.05). Conclusion: Our results indicate that release of nickel ions may affect cell proliferation. The inhibition of cell growth by NiCl2 is mediated by both cell cycle arrest and by induction of cell death. PMID:23198004

Male-induced short oestrous and ovarian cycles in sheep and goats: a working hypothesis.

PubMed

Chemineau, Philippe; Pellicer-Rubio, Maria-Theresa; Lassoued, Narjess; Khaldi, Gley; Monniaux, Danielle

2006-01-01

The existence of short ovulatory cycles (5-day duration) after the first male-induced ovulations in anovulatory ewes and goats, associated or not with the appearance of oestrous behaviour, is the origin of the two-peak abnormal distribution of parturitions after the "male effect". We propose here a working hypothesis to explain the presence of these short cycles. The male-effect is efficient during anoestrus, when follicles contain granulosa cells of lower quality than during the breeding season. They generate corpora lutea (CL) with a lower proportion of large luteal cells compared to small cells, which secrete less progesterone, compared to what is observed in the breeding season cycle. This is probably not sufficient to block prostaglandin synthesis in the endometrial cells of the uterus at the time when the responsiveness to prostaglandins of the new-formed CL is initiated and, in parallel, to centrally reduce LH pulsatility. This LH pulsatility stimulates a new wave of follicles secreting oestradiol which, in turn, stimulates prostaglandin synthesis and provokes luteolysis and new ovulation(s). The occurrence of a new follicular wave on days 3-4 of the first male-induced cycle and the initiation of the responsiveness to prostaglandins of the CL from day 3 of the oestrous cycle are probably the key elements which ensure such regularity in the duration of the short cycles. Exogenous progesterone injection suppresses short cycles, probably not by delaying ovulation time, but rather by blocking prostaglandin synthesis, thus impairing luteolysis. The existence, or not, of oestrous behaviour associated to these ovulatory events mainly varies with species: ewes, compared to does, require a more intense endogenous progesterone priming; only ovulations preceded by normal cycles are associated with oestrous behaviour. Thus, the precise and delicate mechanism underlying the existence of short ovulatory and oestrous cycles induced by the male effect appears to be dependent on the various levels of the hypothalamo-pituitary-ovario-uterine axis.

12-Chloracetyl-PPD, a novel dammarane derivative, shows anti-cancer activity via delay the progression of cell cycle G2/M phase and reactive oxygen species-mediate cell apoptosis.

PubMed

Wang, Xu De; Sun, Yuan Yuan; Zhao, Chen; Qu, Fan Zhi; Zhao, Yu Qing

2017-03-05

(20R)-Dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) is a ginsenoside isolated from Panax ginseng (C. A. Meyer). This compound exhibits anti-cancer activities on many human cancer cell lines. In this study, we investigated anti-cancer mechanisms of 12β-O-( L -Chloracetyl)-dammar-20(22)-ene-3β,25-diol(12-Chloracetyl-PPD), a modified 25-OH-PPD. We found that compound 12-Chloracetyl-PPD resulted in a concentration-dependent inhibition of viability in prostate, breast, and gastric cancer cells, without affecting the viability of normal cell (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2). In MDA-MB-435 and C4-2B cancer cells, 12-Chloracetyl-PPD induced G2/M cell cycle arrest, down-regulated mouse double minute 2 (MDM2) expression, up-regulated p53 expression, triggered apoptosis, and stimulated reactive oxygen species production. Apoptosis can be attenuated by the reactive oxygen species scavenger N-acetylcysteine. Our results suggested that compound 12-Chloracetyl-PPD showed obvious anti-cancer activity based on delaying cell cycle arrest and inducing cell apoptosis by reactive oxygen species production, which supported development of 12-Chloracetyl-PPD as a potential agent for cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

PubMed Central

Stokke, T.; Holte, H.; Smedshammer, L.; Smeland, E. B.; Kaalhus, O.; Steen, H. B.

1998-01-01

We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%). In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03). The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004). When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group (0.8%; P = 0.02). However, there was no significant correlation between the two parameters (P > 0.05). PMID:9667654

Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation

PubMed Central

David, Gregory; Grandinetti, Kathryn B.; Finnerty, Patricia M.; Simpson, Natalie; Chu, Gerald C.; DePinho, Ronald A.

2008-01-01

The Sin3-histone deacetylase (HDAC) corepressor complex is conserved from yeast to humans. Mammals possess two highly related Sin3 proteins, mSin3A and mSin3B, which serve as scaffolds tethering HDAC enzymatic activity, and numerous sequence-specific transcription factors to enable local chromatin regulation at specific gene targets. Despite broad overlapping expression of mSin3A and mSin3B, mSin3A is cell-essential and vital for early embryonic development. Here, genetic disruption of mSin3B reveals a very different phenotype characterized by the survival of cultured cells and lethality at late stages of embryonic development with defective differentiation of multiple lineages—phenotypes that are strikingly reminiscent of those associated with loss of retinoblastoma family members or E2F transcriptional repressors. Additionally, we observe that, whereas mSin3B−/− cells cycle normally under standard growth conditions, they show an impaired ability to exit the cell cycle with limiting growth factors. Correspondingly, mSin3B interacts physically with the promoters of known E2F target genes, and its deficiency is associated with derepression of these gene targets in vivo. Together, these results reveal a critical role for mSin3B in the control of cell cycle exit and terminal differentiation in mammals and establish contrasting roles for the mSin3 proteins in the growth and development of specific lineages. PMID:18332431

Novel pyrimidinic selenourea induces DNA damage, cell cycle arrest, and apoptosis in human breast carcinoma.

PubMed

Barbosa, Flavio A R; Siminski, Tâmila; Canto, Rômulo F S; Almeida, Gabriela M; Mota, Nádia S R S; Ourique, Fabiana; Pedrosa, Rozangela Curi; Braga, Antonio Luiz

2018-06-11

Novel pyrimidinic selenoureas were synthesized and evaluated against tumour and normal cell lines. Among these, the compound named 3j initially showed relevant cytotoxicity and selectivity for tumour cells. Three analogues of 3j were designed and synthesized keeping in view the structural requirements of this compound. Almost all the tested compounds displayed considerable cytotoxicity. However, 8a, one of the 3j analogues, was shown to be highly selective and cytotoxic, especially for breast carcinoma cells (MCF-7) (IC 50  = 3.9 μM). Furthermore, 8a caused DNA damage, inhibited cell proliferation, was able to arrest cell cycle in S phase, and induced cell death by apoptosis in human breast carcinoma cells. Moreover, predictions of pharmacokinetic properties showed that 8a may present good absorption and permeation characteristics for oral administration. Overall, the current study established 8a as a potential drug prototype to be employed as a DNA interactive cytotoxic agent for the treatment of breast cancer. Copyright © 2018. Published by Elsevier Masson SAS.

Cyclin Dependent Kinase Inhibitors as Targets in Ovarian Cancer

DTIC Science & Technology

2005-10-01

STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The objective of this proposal is to develop gene ...have identified key genes that may be effective targets in ovarian cancer therapy. The first three projects seek to identify alterations in these genes ...that allow for high expression of our key gene (s) in ovarian cancer cells but minimal expression in normal tissues. 15. SUBJECT TERMS Cell cycle control

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

Loss of quiescence and self-renewal capacity of hematopoietic stem cell in an in vitro leukemic niche.

PubMed

Vanegas, Natalia-Del Pilar; Vernot, Jean-Paul

2017-01-01

Leukemic and mesenchymal stem cells interact in the leukemic microenvironment and affect each other differently. This interplay has also important implications for the hematopoietic stem cell (HSC) biology and function. This study evaluated human HSC self-renewal potential and quiescence in an in vitro leukemic niche without leukemic cells. A leukemic niche was established by co-culturing mesenchymal stem cells with a fresh conditioned medium obtained from a leukemic (REH) cell line. After 3 days, the REH-conditioned medium was removed and freshly isolated CD34+ at a density of up to 100,000 cells/ml were added to the leukemic niche. CD34+ cell evaluations (cell cycle, self-renewal gene expression and migration capacity) were performed after 3 further days of co-culture. Additionally, we preliminary investigated the soluble factors present in the leukemic niche and their effect on the mesenchymal stem cells. Statistical significance was assessed by Student's t test or the nonparametric test Kolmogorov-Smirnov. By co-culturing normal mesenchymal stem cells with the REH-conditioned medium we showed that hematopoietic stem cells, normally in a quiescent state, enter cell cycle and proliferate. This loss of quiescence was accompanied by an increased expression of Ki-67 and c-Myc, two well-known cell proliferation-associated markers. Two central regulators of quiescence GATA2 and p53 were also down regulated. Importantly, two genes involved in HSC self-renewal, Klf4 and the histone-lysine N -methyltransferase enzyme Ezh2, were severely affected. On the contrary, c-Kit expression, the stem cell factor receptor, was upregulated in hematopoietic stem cells when compared to the normal niche. Interestingly, mesenchymal stem cells incubated with the REH-conditioned medium stopped growing, showed a flattened morphology with the appearance of small vacuoles, and importantly, became positive for the senescence-associated beta-galactosidase activity. Evaluation of the leukemic-conditioned medium showed increased IL-6 and IL-8, suggesting that these cytokines could be responsible for the observed changes. Our results showed that quiescence and self-renewal are severely affected in this leukemic niche. This in vitro leukemic niche, established without leukemic cells, will facilitate HSC gene expression evaluation and the development of therapeutic agents aimed to neutralize soluble factors and the cell signaling pathways involved in HSC alterations.

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

PubMed

Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

2015-08-01

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression. © 2015 Wiley Periodicals, Inc.

TESTIN Induces Rapid Death and Suppresses Proliferation in Childhood B Acute Lymphoblastic Leukaemia Cells

PubMed Central

Weeks, Robert J.; Ludgate, Jackie L.; LeMée, Gwenn; Morison, Ian M.

2016-01-01

Background Childhood acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Despite high cure rates, side effects and late consequences of the intensive treatments are common. Unquestionably, the identification of new therapeutic targets will lead to safer, more effective treatments. We identified TES promoter methylation and transcriptional silencing as a very common molecular abnormality in childhood ALL, irrespective of molecular subtype. The aims of the present study were to demonstrate that TES promoter methylation is aberrant, to determine the effects of TES re-expression in ALL, and to determine if those effects are mediated via TP53 activity. Methods Normal fetal and adult tissue DNA was isolated and TES promoter methylation determined by Sequenom MassARRAY. Quantitative RT-PCR and immunoblot were used to confirm re-expression of TES in ALL cell lines after 5’-aza-2’-deoxycytidine (decitabine) exposure or transfection with TES expression plasmids. The effects of TES re-expression on ALL cells were investigated using standard cell proliferation, cell death and cell cycle assays. Results In this study, we confirm that the TES promoter is unmethylated in normal adult and fetal tissues. We report that decitabine treatment of ALL cell lines results in demethylation of the TES promoter and attendant expression of TES mRNA. Re-expression of TESTIN protein in ALL cells using expression plasmid transfection results in rapid cell death or cell cycle arrest independent of TP53 activity. Conclusions These results suggest that TES is aberrantly methylated in ALL and that re-expression of TESTIN has anti-leukaemia effects which point to novel therapeutic opportunities for childhood ALL. PMID:26985820

Successful resection of a giant mediastinal non-seminomatous germ cell tumor showing fluorodeoxyglucose accumulation after neoadjuvant chemotherapy: report of a case.

PubMed

Takada, Kazuki; Morodomi, Yosuke; Okamoto, Tatsuro; Suzuki, Yuzo; Fujishita, Takatoshi; Kitahara, Hirokazu; Shimamatsu, Shinichiro; Kohno, Mikihiro; Kawano, Daigo; Hidaka, Noriko; Nakanishi, Yoichi; Maehara, Yoshihiko

2014-05-01

A 32-year-old man presented with a mediastinal non-seminomatous germ cell tumor showing fluorodeoxyglucose (FDG) accumulation (maximum standardized uptake value = 22.21) and extremely elevated blood alpha-fetoprotein (AFP) level (9203.0 ng/ml). The patient underwent 4 cycles of neoadjuvant chemotherapy (cisplatin, bleomycin, and etoposide), which normalized the AFP level and reduced the tumor size, allowing complete resection without a support of extracorporeal circulation. Despite preoperative positron emission tomography revealing increased FDG uptake in the residual tumor (maximum standardized uptake value = 3.59), the pathologic evaluation revealed that no viable germ cell tumor cells remained. We believe FDG uptake should not be used as a criterion for surgical resection after neoadjuvant chemotherapy. It is appropriate to resect the residual tumor regardless of FDG uptake after induction chemotherapy if a tumor is resectable and the AFP level normalizes.

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

PubMed

Thuraisamy, Thujitha; Lodato, Patricia B

2018-05-01

In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

The DREAM complex through its subunit Lin37 cooperates with Rb to initiate quiescence

PubMed Central

Mages, Christina FS; Wintsche, Axel; Bernhart, Stephan H

2017-01-01

The retinoblastoma Rb protein is an important factor controlling the cell cycle. Yet, mammalian cells carrying Rb deletions are still able to arrest under growth-limiting conditions. The Rb-related proteins p107 and p130, which are components of the DREAM complex, had been suggested to be responsible for a continued ability to arrest by inhibiting E2f activity and by recruiting chromatin-modifying enzymes. Here, we show that p130 and p107 are not sufficient for DREAM-dependent repression. We identify the MuvB protein Lin37 as an essential factor for DREAM function. Cells not expressing Lin37 proliferate normally, but DREAM completely loses its ability to repress genes in G0/G1 while all remaining subunits, including p130/p107, still bind to target gene promoters. Furthermore, cells lacking both Rb and Lin37 are incapable of exiting the cell cycle. Thus, Lin37 is an essential component of DREAM that cooperates with Rb to induce quiescence. PMID:28920576

p21(WAF1) Mediates Cell-Cycle Inhibition, Relevant to Cancer Suppression and Therapy.

PubMed

El-Deiry, Wafik S

2016-09-15

p21 (WAF1/CIP1; CDKN1a) is a universal cell-cycle inhibitor directly controlled by p53 and p53-independent pathways. Knowledge of the regulation and function of p21 in normal and cancer cells has opened up several areas of investigation and has led to novel therapeutic strategies. The discovery in 1993 and subsequent work on p21 has illuminated basic cellular growth control, stem cell phenotypes, the physiology of differentiation, as well as how cells respond to stress. There remain open questions in the signaling networks, the ultimate role of p21 in the p53-deficiency phenotype in the context of other p53 target defects, and therapeutic strategies continue to be a work in progress. Cancer Res; 76(18); 5189-91. ©2016 AACRSee related article by El-Deiry et al., Cancer Res 1994;54:1169-74Visit the Cancer Research 75(th) Anniversary timeline. ©2016 American Association for Cancer Research.

Pre-exposure to 50 Hz-electromagnetic fields enhanced the antiproliferative efficacy of 5-fluorouracil in breast cancer MCF-7 cells

PubMed Central

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

2018-01-01

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

β1 integrin is a crucial regulator of pancreatic β-cell expansion

PubMed Central

Diaferia, Giuseppe R.; Jimenez-Caliani, Antonio J.; Ranjitkar, Prerana; Yang, Wendy; Hardiman, Gary; Rhodes, Christopher J.; Crisa, Laura; Cirulli, Vincenzo

2013-01-01

Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the β1 integrin gene in developing pancreatic β-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking β1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of β-cells to only ∼18% of wild-type levels. Despite the significant reduction in β-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of β-cells lacking β1 integrin revealed a normal expression repertoire of β-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in β-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that β1 integrin receptors function as crucial positive regulators of β-cell expansion. PMID:23863477

Development of mice without Cip/Kip CDK inhibitors.

PubMed

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu; Hara, Eiji; Nakayama, Keiko; Nakayama, Keiichi I

2012-10-19

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage). Copyright © 2012 Elsevier Inc. All rights reserved.

Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

PubMed Central

Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

2011-01-01

Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability

PubMed Central

Pozo, Pedro N.; Cook, Jeanette Gowen

2016-01-01

Successful cell proliferation requires efficient and precise genome duplication followed by accurate chromosome segregation. The Cdc10-dependent transcript 1 protein (Cdt1) is required for the first step in DNA replication, and in human cells Cdt1 is also required during mitosis. Tight cell cycle controls over Cdt1 abundance and activity are critical to normal development and genome stability. We review here recent advances in elucidating Cdt1 molecular functions in both origin licensing and kinetochore–microtubule attachment, and we describe the current understanding of human Cdt1 regulation. PMID:28025526

Organization of supercoil domains and their reorganization by transcription

PubMed Central

Deng, Shuang; Stein, Richard A.; Higgins, N. Patrick

2006-01-01

Summary During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems. PMID:16135220

Development of mice without Cip/Kip CDK inhibitors

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

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu

2012-10-19

Highlights: Black-Right-Pointing-Pointer Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. Black-Right-Pointing-Pointer Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. Black-Right-Pointing-Pointer CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largelymore » unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G{sub 0} to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage).« less

Promoter methylation patterns in Richter syndrome affect stem-cell maintenance and cell cycle regulation and differ from de novo diffuse large B-cell lymphoma.

PubMed

Rinaldi, Andrea; Mensah, Afua Adjeiwaa; Kwee, Ivo; Forconi, Francesco; Orlandi, Ester M; Lucioni, Marco; Gattei, Valter; Marasca, Roberto; Berger, Françoise; Cogliatti, Sergio; Cavalli, Franco; Zucca, Emanuele; Gaidano, Gianluca; Rossi, Davide; Bertoni, Francesco

2013-10-01

In a fraction of patients, chronic lymphocytic leukaemia (CLL) can transform to Richter syndrome (RS), usually a diffuse large B-cell lymphoma (DLBCL). We studied genome-wide promoter DNA methylation in RS and clonally related CLL-phases of transformed patients, alongside de novo DLBCL (of non-germinal centre B type), untransformed-CLL and normal B-cells. The greatest differences in global DNA methylation levels were observed between RS and DLBCL, indicating that these two diseases, although histologically similar, are epigenetically distinct. RS was more highly methylated for genes involved in cell cycle regulation. When RS was compared to the preceding CLL-phase and with untransformed-CLL, RS presented a higher degree of methylation for genes possessing the H3K27me3 mark and PRC2 targets, as well as for gene targets of TP53 and RB1. Comparison of the methylation levels of individual genes revealed that OSM, a stem cell regulatory gene, exhibited significantly higher methylation levels in RS compared to CLL-phases. Its transcriptional repression by DNA methylation was confirmed by 5-aza-2'deoxycytidine treatment of DLBCL cells, determining an increased OSM expression. Our results showed that methylation patterns in RS are largely different from de novo DLBCL. Stem cell-related genes and cell cycle regulation genes are targets of DNA methylation in RS. © 2013 John Wiley & Sons Ltd.

The Trophic Life Cycle Stage of the Opportunistic Fungal Pathogen Pneumocystis murina Hinders the Ability of Dendritic Cells To Stimulate CD4+ T Cell Responses

PubMed Central

Evans, Heather M.; Simpson, Andrew; Shen, Shu; Stromberg, Arnold J.; Pickett, Carol L.

2017-01-01

ABSTRACT The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including β-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with β-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells. PMID:28694293

Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes.

PubMed

Clare, Susan E; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z; Kim, J Julie; Khan, Seema A

2016-05-23

The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.

Novel anticancer agent, benzyldihydroxyoctenone, isolated from Streptomyces sp. causes G1 cell cycle arrest and induces apoptosis of HeLa cells.

PubMed

Lee, Chul-Hoon; Lim, Haeyoung; Moon, Sangik; Shin, Choonshik; Kim, Seunghyun; Kim, Bum-Joon; Lim, Yoongho

2007-06-01

In the course of screening for anticancer agents, a novel active compound, F3-2-5, was isolated from culture broth of Streptomyces sp., KACC91015. Its structure was identified using nuclear magnetic resonance, mass spectrometry, and molecular modeling experiments, and confirmed by total synthesis. The growth of various human cancer cell lines was inhibited in a dose-dependent manner by 0.06-0.48 mM F3-2-5 over 24 h. Its IC(50) values were estimated at 37 microM on HeLa, 72 microM on A549, and 190 microM on HT-29 cells. However, F3-2-5 had no antiproliferative effect on normal lymphocytes and normal fibroblasts used as controls. Moreover, it affected cell cycle regulation and caused apoptosis of the HeLa cells; chromatin condensation and DNA fragmentation were observed in cells exposed to 80 microM F3-2-5. Western blot analysis revealed that F3-2-5 inhibited phosphorylation of retinoblastoma protein (pRb) and reduced expression of cyclin-dependent kinase-4 and -6, and cyclin D1 and E, while levels of p53 and p21(WAF1/CIP1) increased. Taken together, these findings show that F3-2-5 inhibits proliferation of HeLa cells by inducing G(1) phase arrest as a consequence of inhibition of pRb phosphorylation following up-regulation of p21(WAF1/CIP1) and p53. Furthermore, apoptosis in HeLa cells treated with F3-2-5 was associated with an increase in Bax and p53, leading to release of cytochrome c, activation of caspase-3, and -8, and cleavage of poly (ADP-ribose) polymerase.

cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

NASA Astrophysics Data System (ADS)

Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

2017-06-01

Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

A large shRNA library approach identifies lncRNA Ntep as an essential regulator of cell proliferation

PubMed Central

Beermann, Julia; Kirste, Dominique; Iwanov, Katharina; Lu, Dongchao; Kleemiß, Felix; Kumarswamy, Regalla; Schimmel, Katharina; Bär, Christian; Thum, Thomas

2018-01-01

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach. PMID:29099486

Is whole-culture synchronization biology's 'perpetual-motion machine'?

PubMed

Cooper, Stephen

2004-06-01

Whole-culture or batch synchronization cannot, in theory, produce a synchronized culture because it violates a fundamental law that proposes that no batch treatment can alter the cell-age order of a culture. In analogy with the history of perpetual-motion machines, it is suggested that the study of these whole-culture 'synchronization' methods might lead to an understanding of general biological principles even though these methods cannot be used to study the normal cell cycle.

Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

PubMed

Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

2014-03-01

Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

PubMed Central

McDade, Simon S.; Patel, Daksha; Moran, Michael; Campbell, James; Fenwick, Kerry; Kozarewa, Iwanka; Orr, Nicholas J.; Lord, Christopher J.; Ashworth, Alan A.; McCance, Dennis J.

2014-01-01

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair. PMID:24823795

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

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

Chen, Yi; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

2013-09-15

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cellmore » proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.« less

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1986-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

Clonal evolution following chemotherapy-induced stem cell depletion in cats heterozygous for glucose-6-phosphate dehydrogenase

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

Abkowitz, J.L.; Ott, R.M.; Holly, R.D.

The number of hematopoietic stem cells necessary to support normal hematopoiesis is not known but may be small. If so, the depletion or damage of such cells could result in apparent clonal dominance. To test this hypothesis, dimethylbusulfan (2 to 4 mg/kg intravenously (IV) x 3) was given to cats heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD). These cats were the daughters of domestic X Geoffroy parents. After the initial drug-induced cytopenias (2 to 4 weeks), peripheral blood counts and the numbers of marrow progenitors detected in culture remained normal, although the percentages of erythroid burst-forming cells (BFU-E) andmore » granulocyte/macrophage colony-forming cells (CFU-GM) in DNA synthesis increased, as determined by the tritiated thymidine suicide technique. In three of six cats treated, a dominance of Geoffroy-type G-6-PD emerged among the progenitor cells, granulocytes, and RBCs. These skewed ratios of domestic to Geoffroy-type G-6-PD have persisted greater than 3 years. No changes in cell cycle kinetics or G-6-PD phenotypes were noted in similar studies in six control cats. These data suggest that clonal evolution may reflect the depletion or damage of normal stem cells and not only the preferential growth and dominance of neoplastic cells.« less

Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

PubMed

Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

2016-12-01

A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC 50 : 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC 50 : 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC 50 : 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dual overcharge protection and solid electrolyte interphase-improving action in Li-ion cells containing a bis-annulated dialkoxyarene electrolyte additive

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Shkrob, Ilya A.; Assary, Rajeev S.; Zhang, Shuo; Hu, Bin; Liao, Chen; Zhang, Zhengcheng; Zhang, Lu

2018-02-01

1,4-Dialkoxybenzene additives are commonly used as redox active shuttles in lithium-ion batteries in order to prevent runaway oxidation of electrolyte when overcharge conditions set in. During this action the shuttle molecule goes through a futile cycle, becoming oxidized at the cathode and reduced at the anode. Minimizing parasitic reactions in all states of charge is paramount for sustained protective action. Here we demonstrate that recently developed bis-annulated 9,10-bis(2-methoxyethoxy)-1,2,3,4,5,6,7,8-octahydro-1,4:5,8-dimethano-anthracene shuttle molecule (that yields exceptionally stable radical cations) survives over 120 cycles of overcharge abuse with 100% overcharge ratio at C/5 rate. Equally remarkably, in the presence of this additive the cell impedance becomes significantly lower compared to the control cells without the additive; this decrease is observed during the formation, normal cycling, and even under overcharge conditions. This unusual dual action has not been observed in other redox shuttle systems, and it presents considerable practical interest.

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat

PubMed Central

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan

2014-01-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome. PMID:25285307

Temperature oscillations drive cycles in the activity of MMP-2,9 secreted by a human trabecular meshwork cell line.

PubMed

Li, Stanley Ka-Lok; Banerjee, Juni; Jang, Christopher; Sehgal, Amita; Stone, Richard A; Civan, Mortimer M

2015-02-05

Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

In vitro short-term exposure to air pollution PM{sub 2.5-0.3} induced cell cycle alterations and genetic instability in a human lung cell coculture model

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

Abbas, Imane; EA4492-UCEIV, Université du Littoral-Côte d’Opale, Dunkerque; Lebanese Atomic Energy Commission – CNRS, Beirut

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOHmore » and/or MSI) in the PM{sub 2.5-0.3}-exposed coculture model. PM{sub 2.5-0.3} exposure of human AM from the coculture model induced marked cell cycle alterations after 24 h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM{sub 2.5-0.3} was reported in the L132 cells. Exposure of human AM from the coculture model to PM{sub 2.5-0.3} resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM{sub 2.5-0.3} induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability. - Highlights: • Better knowledge on health adverse effects of air pollution PM{sub 2.5}. • Human alveolar macrophage and normal human epithelial lung cell coculture. • Molecular abnormalities from TP53-RB gene signaling pathway. • Loss of heterozygosity and microsatellite instability. • Pathologic changes in morphology and number of cells in relation to airway remodeling.« less

Bax Translocation Mediated Mitochondrial Apoptosis and Caspase Dependent Photosensitizing Effect of Ficus religiosa on Cancer Cells

PubMed Central

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

2012-01-01

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

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas.

PubMed

Thompson, Elizabeth A; Zhu, Songyun; Hall, Jonathan R; House, John S; Ranjan, Rakesh; Burr, Jeanne A; He, Yu-Ying; Owens, David M; Smart, Robert C

2011-06-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.

C/EBPα Expression Is Downregulated in Human Nonmelanoma Skin Cancers and Inactivation of C/EBPα Confers Susceptibility to UVB-Induced Skin Squamous Cell Carcinomas

PubMed Central

Thompson, Elizabeth A.; Zhu, Songyun; Hall, Jonathan R.; House, John S.; Ranjan, Rakesh; Burr, Jeanne A.; He, Yu-Ying; Owens, David M.; Smart, Robert C.

2012-01-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. PMID:21346772

Effect on intensity of treadmill running on learning, memory and expressions of cell cycle-related proteins in rats with cerebral ischemia.

PubMed

Zhao, Ya-Ning; Li, Jian-Min; Chen, Chang-Xiang; Li, Shu-Xing; Xue, Cheng-Jing

2017-06-20

We discussed the intensity of treadmill running on learning, memory and expression of cell cycle-related proteins in rats with cerebral ischemia. Eighty healthy male SD rats were randomly divided into normal group, model group, intensity I group and intensity II group, with 20 rats in each group. The four-vessel occlusion method of Pulsinelli (4-VO) was used to induce global cerebral ischemia. Brain neuronal morphology was observed by hematoxylin-eosin (HE) staining at 3h, 6h, 24h and 48h after modeling, respectively. Hippocampal expressions of cyclin A and cyclin E were detected by immunohistochemistry. At 48h after modeling, the learning and memory performance of rats was tested by water maze experiment. Compared with the normal group, the other three groups had a significant reduction in surviving neurons, prolonging of escape latency and decreased number of passes over the former position of the platform (P<0.05). The number of surviving neurons and the number of passes over the former position of the platform were obviously lower in the model group than in intensity I group (P<0.05), but significantly higher compared with intensity II group (P<0.05). Escape latency of the model group was obviously prolonged as compared with intensity I group (P<0.05), but much shorter than that of intensity II group (P<0.05). Compared with the normal group, the expressions of cyclin A and cyclin E were significantly upregulated at different time points after modeling (P<0.05). The expression of the model group was higher than that of intensity I group, but lower than that of intensity II group (P<0.05). Moderate intensity of treadmill running can help protect brain neurons and improve learning and memory performance of rats with global cerebral ischemia. But high intensity of treadmill running has a negative impact, possibly through the regulation of cell cycle-related proteins in ischemia/reperfusion injury.

Identification of novel inhibitors of human Chk1 using pharmacophore-based virtual screening and their evaluation as potential anti-cancer agents

NASA Astrophysics Data System (ADS)

Kumar, Vikash; Khan, Saman; Gupta, Priyanka; Rastogi, Namrata; Mishra, Durga Prasad; Ahmed, Shakil; Siddiqi, Mohammad Imran

2014-12-01

Kinases are one of the major players in cancer development and progression. Serine threonine kinases such as human checkpoint kinase-1 (Chk1), Mek1 and cyclin-dependent kinases have been identified as promising targets for cancer treatment. Chk1 is an important kinase with vital role in cell cycle arrest and many potent inhibitors targeted to Chk1 have been reported and few are currently in clinical trials. Considering the emerging importance of Chk1 inhibitors in cancer treatment there is a need to widen the chemical space of Chk1 inhibitors. In this study, we are reporting an integrated in silico approach to identify novel competitive Chk1 inhibitors. A 4-features pharmacophore model was derived from a co-crystallized structure of known potent Chk1 inhibitor and subjected to screen Maybridge compound library. Hits obtained from the screening were docked into the Chk1 active site and filtered on the basis of docking score and the number of pharmacophoric features showing conserved interaction within the active site of Chk1. Further, five compounds from the top ranking hits were subjected to in vitro evaluation as Chk1 inhibitor. After the kinase assay, four compounds were found to be active against human Chk1 (IC50 range from 4.2 to 12.5 µM). Subsequent study using the cdc25-22 mutant yeast cells revealed that one of compound (SPB07479; IC50 = 4.24 µM) promoted the formation of multinucleated cells, therefore overriding the cell cycle checkpoint. Validation studies using normal and human cancer cell lines, indicated that SPB07479 significantly inhibited proliferation of cervical cancer cells as a single agent and chemosensitized glioma and pancreatic cancer cell lines to standard chemotherapy while sparing normal cells. Additionally SPB07479 did not show significant cytotoxicity in normal cells. In conclusion we report that SPB07479 appear promising for further development of Chk1 inhibitors. This study also highlights the role of conserved water molecules in the active site of Chk1 for the successful identification of novel inhibitors.

Downregulation of HDAC9 inhibits cell proliferation and tumor formation by inducing cell cycle arrest in retinoblastoma

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

Zhang, Yiting; Wu, Dan; Xia, Fengjie

Histone deacetylase 9 (HDAC9) is a member of class II HDACs, which regulates a wide variety of normal and abnormal physiological functions. Recently, HDAC9 has been found to be overexpressed in some types of human cancers. However, the role of HDAC9 in retinoblastoma remains unclear. In this study, we found that HDAC9 was commonly expressed in retinoblastoma tissues and HDAC9 was overexpressed in prognostically poor retinoblastoma patients. Through knocking down HDAC9 in Y79 and WERI-Rb-1 cells, the expression level of HDAC9 was found to be positively related to cell proliferation in vitro. Further investigation indicated that knockdown HDAC9 could significantly induce cellmore » cycle arrest at G1 phase in retinoblastoma cells. Western blot assay showed downregulation of HDAC9 could significantly decrease cyclin E2 and CDK2 expression. Lastly, xenograft study in nude mice showed that downregulation of HDAC9 inhibited tumor growth and development in vivo. Therefore, our results suggest that HDAC9 could serve as a novel potential therapeutic target in the treatment of retinoblastoma. - Highlights: • High expression of HDAC9 correlates with poor patient prognosis. • Downregulation of HDAC9 inhibits cell proliferation in retinoblastoma cells. • Downregulation of HDAC9 induces cell cycle arrest at G1 phase in retinoblastoma cells. • Downregulation of HDAC9 suppresses tumor growth in nude mice.« less

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

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

2009-03-01

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

Cyclin D1-Cdk4 controls glucose metabolism independently of cell cycle progression.

PubMed

Lee, Yoonjin; Dominy, John E; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I; Puigserver, Pere

2014-06-26

Insulin constitutes a principal evolutionarily conserved hormonal axis for maintaining glucose homeostasis; dysregulation of this axis causes diabetes. PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) links insulin signalling to the expression of glucose and lipid metabolic genes. The histone acetyltransferase GCN5 (general control non-repressed protein 5) acetylates PGC-1α and suppresses its transcriptional activity, whereas sirtuin 1 deacetylates and activates PGC-1α. Although insulin is a mitogenic signal in proliferative cells, whether components of the cell cycle machinery contribute to its metabolic action is poorly understood. Here we report that in mice insulin activates cyclin D1-cyclin-dependent kinase 4 (Cdk4), which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high-throughput chemical screen, we identify a Cdk4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK-3β (glycogen synthase kinase 3-beta) signalling induces cyclin D1 protein stability by sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 messenger RNA transcripts. Activated cyclin D1-Cdk4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycaemia. In diabetic models, cyclin D1-Cdk4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycaemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division.

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

PubMed

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

2017-06-01

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

P16INK4a MEDIATED SUPPRESSION OF TELOMERASE IN NORMAL AND MALIGNANT HUMAN BREAST CELLS

PubMed Central

Bazarov, Alexey V.; van Sluis, Marjolein; Hines, Curtis; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W.; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-01-01

Summary The cyclin-dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. PMID:20569236

Myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway

PubMed Central

HUANG, HAIZHI; CHEN, ALLEN Y.; YE, XINGQIAN; LI, BINGYUN; ROJANASAKUL, YON; RANKIN, GARY O.; CHEN, YI CHARLIE

2015-01-01

Cisplatin is a commonly used drug for cancer treatment by crosslinking DNA, leading to apoptosis of cancer cells, resistance to cisplatin treatment often occurs, leading to relapse. Therefore, there is a need for the development of more effective treatment strategies that can overcome chemoresistance. Myricetin is a flavonoid from fruits and vegetables, showing anticancer activity in various cancer cells. In this study, we found myricetin exhibited greater cytotoxicity than cisplatin in two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and it was less cytotoxic to the normal ovarian cell line IOSE-364. Myricetin selectively induced apoptosis in both cisplatin-resistant cancer cell lines, but did not induce apoptosis in the normal ovarian cell line. It induced both Bcl-2 family-dependent intrinsic and DR5 dependent extrinsic apoptosis in OVCAR-3 cells. P53, a multifunctional tumor suppressor, regulated apoptosis in OVCAR-3 cells through a Bcl-2 family protein-dependent pathway. Myricetin did not induce cell cycle arrest in either ovarian cancer cell line. Because of its potency and selectivity against cisplatin-resistant cancer cells, myricetin could potentially be used to overcome cancer chemoresistance against platinum-based therapy. PMID:26315556

Abnormal Uterine Bleeding FAQ

MedlinePlus

... Abnormal Uterine Bleeding • What is a normal menstrual cycle? • When is bleeding abnormal? • At what ages is ... abnormal bleeding? •Glossary What is a normal menstrual cycle? The normal length of the menstrual cycle is ...

Smooth muscle membrane organization in the normal and dysfunctional human urinary bladder: a structural analysis.

PubMed

Burkhard, Fiona C; Monastyrskaya, Katia; Studer, Urs E; Draeger, Annette

2005-01-01

The decline in contractile properties is a characteristic feature of the dysfunctional bladder as a result of infravesical outlet obstruction. During clinical progression of the disease, smooth muscle cells undergo structural modifications. Since adaptations to constant changes in length require a high degree of structural organization within the sarcolemma, we have investigated the expression of several proteins, which are involved in smooth muscle membrane organization, in specimens derived from normal and dysfunctional organs. Specimen from patients with urodynamically normal/equivocal (n = 4), obstructed (n = 2), and acontractile (n = 2) bladders were analyzed relative to their structural features and sarcolemmal protein profile. Smooth muscle cells within the normal urinary bladder display a distinct sarcolemmal domain structure, characterized by firm actin-attachment sites, alternating with flexible "hinge" regions. In obstructed bladders, foci of cells displaying degenerative sarcolemmal changes alternate with areas of hypertrophic cells in which the membrane appears unaffected. In acontractile organs, the overall membrane structure remains intact, however annexin 6, a protein belonging to a family of Ca2+-dependent, "membrane-organizers," is downregulated. Degenerative changes in smooth muscle cells, which are chronically working against high resistance, are preferentially located within the actin-attachment sites. In acontractile bladders, the downregulation of annexin 6 might have a bearing on the fine-tuning of the plasma membrane during contraction/relaxation cycles. Copyright 2005 Wiley-Liss, Inc.

Dynamics of infection with human herpesvirus-6 in EBV-negative infectious mononucleosis: data acquisition for computer modeling.

PubMed

Krueger, G R; Bertram, G; Ramon, A; Koch, B; Ablashi, D V; Brandt, M E; Wang, G; Buja, L M

2001-01-01

Ten adult patients with active HHV-6 variant A infections and clinical infectious mononucleosis-like disease (IM) were studied over a period of 32 weeks after onset of disease for their viral DNA load, changes in peripheral blood T-lymphocytes and subpopulations and frequency of cell death in peripheral blood cells. The data were collected as the basis for an advanced computer simulation study for which available data in the literature were too varied. Since the exact time of primary infection of the patients was not known and thus no time relationship of viral effects at cellular level were determined, we supplemented such data from separate tissue culture studies using HHV-6 alpha infection of HSB2 cells. Patients with IM demonstrate an increase in-HHV-6 DNA copies from 0 to 8.2 log 10/5 microL blood within 4 weeks return to normal by 16 weeks. Total T-lymphocytes follow infection with a 20-fold increase above normal peaking at 8-10 weeks and then return to normal by 24-28 weeks. Coincidently, less mature lymphoid cells carrying markers for stem cells, thymic cortical and medullary cells increase 8-10-fold indicating an enhanced mobilization of such cells from premature cell compartments. Cell death in peripheral mononuclear cells peaked with 30% at 8 weeks after onset of clinical disease and normalized by 24 weeks. HHV-6 replication in cell culture as determined by antigen expression, electron microscopy and harvest of infectious virus indicated a complete cycle of virus infection and replication of at least 6 days. The presented data compare well with others from the literature and will serve for testing in a computer simulation model, which is the subject of a forthcoming paper.

cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

PubMed Central

Shi, Ting; Mazumdar, Tapati; DeVecchio, Jennifer; Duan, Zhong-Hui; Agyeman, Akwasi; Aziz, Mohammad; Houghton, Janet A.

2010-01-01

Background Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. Methodology/Principal Findings To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. Conclusions/Significance This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary. PMID:20957031

In vitro genotoxicity of exhaust emissions of diesel and gasoline engine vehicles operated on a unified driving cycle.

PubMed

Liu, Yu-Qing; Keane, Michael; Ensell, Mang; Miller, William; Kashon, Michael; Ong, Tong-man; Mauderly, Joe; Lawson, Doug; Gautam, Mridul; Zielinska, Barbara; Whitney, Kevin; Eberhardt, James; Wallace, William

2005-01-01

Acetone extracts of engine exhaust particulate matter (PM) and of vapor-phase semi-volatile organic compounds (SVOCs) collected from a set of 1998-2000 model year normal emitter diesel engine automobile or light trucks and from a set of 1982-1996 normal emitter gasoline engine automobiles or light trucks operated on the California Unified Driving Cycle at 22 [degree]C were assayed for in vitro genotoxic activities. Gasoline and diesel PM were comparably positive mutagens for Salmonella typhimurium strains YG1024 and YG1029 on a mass of PM extract basis with diesel higher on a mileage basis; gasoline SVOC was more active than diesel on an extracted-mass basis, with diesel SVOC more active on a mileage basis. For chromosomal damage indicated by micronucleus induction in Chinese hamster lung fibroblasts (V79 cells), diesel PM expressed about one-tenth that of gasoline PM on a mass of extract basis, but was comparably active on a mileage basis; diesel SVOC was inactive. For DNA damage in V79 cells indicated by the single cell gel electrophoresis (SCGE) assay, gasoline PM was positive while diesel PM was active at the higher doses; gasoline SVOC was active with toxicity preventing measurement at high doses, while diesel SVOC was inactive at all but the highest dose.

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells.

PubMed

Kruitwagen, Hedwig S; Westendorp, Bart; Viebahn, Cornelia S; Post, Krista; van Wolferen, Monique E; Oosterhoff, Loes A; Egan, David A; Delabar, Jean-Maurice; Toussaint, Mathilda J; Schotanus, Baukje A; de Bruin, Alain; Rothuizen, Jan; Penning, Louis C; Spee, Bart

2018-01-15

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.

Correlation and comparison of Nb/sub 2/ lymphoma cell bioassay with radioimmunoassay for human prolactin

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

Subramanian, M.G.; Spirtos, N.J.; Moghissi, K.S.

Serum samples from groups of men and women with normal and elevated prolactin (PRL) levels were assayed by radioimmunoassay (RIA) and by Nb/sub 2/ lymphoma cell bioassay (BA) for the presence of PRL. Because the Nb/sub 2/ lymphoma cells respond to both PRL and growth hormone, BA for PRL activity was carried out before and after neutralization of growth hormone in the serum samples. There were excellent correlations between RIA and BA both in euprolactinemic and hyperprolactinemic subjects. On an absolute basis, RIA and BA values were similar in the euprolactinemic group (6.6 +/- 0.8 versus 6.2 +/- 1.0), whereasmore » in the hyperprolactinemic group, RIA values were significantly higher than the BA results. The two assay systems also appeared to correlate better in women who were hyperprolactinemic, with obvious menstrual cycle disturbances, than in hyperprolactinemic women without menstrual cycle disturbances.« less

DEVELOPMENT OF THE METHOD AND U.S. NORMALIZATION DATABASE FOR LIFE CYCLE IMPACT ASSESSMENT AND SUSTAINABILITY METRICS

EPA Science Inventory

Normalization is an optional step within Life Cycle Impact Assessment (LCIA) that may be used to assist in the interpretation of life cycle inventory data as well as, life cycle impact assessment results. Normalization transforms the magnitude of LCI and LCIA results into relati...

Alternol induces an S-phase arrest of melanoma B16F0 cells.

PubMed

Liu, Liangliang; Zhang, Bo; Yuan, Xuan; Wang, Penglong; Sun, Xiling; Zheng, Qiusheng

2014-03-01

Alternol is a novel compound purified from the fermentation products of a microorganism in the yew tree bark. This study looks at the effects of alternol on the proliferation and cell cycle distribution of mouse melanoma cells. The inhibition of cell proliferation and changes in cell cycle distribution were analysed by sulforhodamine B and flow cytometry assays, respectively. mRNA expression of cyclin A, cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) and CDK inhibitor1A (p21) were measured by real-time reverse transcription PCR (RT-PCR). The protein levels of cyclin A, CDK2 and PCNA were analysed by Western blot analysis. p21 was measured by ELISA. Alternol treatment caused a significant decrease in the proliferation rate of B16F0 and B16F10 cells, which were significantly arrested in S phase, but this treatment had less effect on normal human embryonic kidney 293T cells. The mechanism by which alternol inhibits B16F0 proliferation in vitro may be associated with the inhibition of CDK2 and PCNA, and the activation of p21. © 2013 International Federation for Cell Biology.

Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression

PubMed Central

Audette, Dylan S.; Anand, Deepti; So, Tammy; Rubenstein, Troy B.; Lachke, Salil A.; Lovicu, Frank J.; Duncan, Melinda K.

2016-01-01

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF. PMID:26657765

Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression.

PubMed

Audette, Dylan S; Anand, Deepti; So, Tammy; Rubenstein, Troy B; Lachke, Salil A; Lovicu, Frank J; Duncan, Melinda K

2016-01-15

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF. © 2016. Published by The Company of Biologists Ltd.

Dosage-Sensitive Function of RETINOBLASTOMA RELATED and Convergent Epigenetic Control Are Required during the Arabidopsis Life Cycle

PubMed Central

Johnston, Amal J.; Kirioukhova, Olga; Barrell, Philippa J.; Rutten, Twan; Moore, James M.; Baskar, Ramamurthy; Grossniklaus, Ueli; Gruissem, Wilhelm

2010-01-01

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development. PMID:20585548

Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

PubMed

Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

2016-05-01

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture between the ESCC and normal epithelial tissues. However, there were no differences observed between the young and old patients. © 2015 International Society for Diseases of the Esophagus.

The influence of high glucose on the Cip/Kip family expression profiles in HRECs.

PubMed

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

2013-12-01

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

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

PubMed Central

Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

2012-01-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

2012-07-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells.

PubMed

Kang, Minkyung; Jeong, Wooyoung; Bae, Hyocheol; Lim, Whasun; Bazer, Fuller W; Song, Gwonhwa

2018-03-01

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals. © 2017 Wiley Periodicals, Inc.

A Futile Redox Cycle Involving Neuroglobin Observed at Physiological Temperature.

PubMed

Liu, Anyang; Brittain, Thomas

2015-08-24

Previous studies identifying the potential anti-apoptotic role of neuroglobin raise the question as to how cells might employ neuroglobin to avoid the apoptotic impact of acute hypoxia whilst also avoiding chronic enhancement of tumour formation. We show that under likely physiological conditions neuroglobin can take part in a futile redox cycle. Determination of the rate constants for each of the steps in the cycle allows us to mathematically model the steady state concentration of the active anti-apoptotic ferrous form of neuroglobin under various conditions. Under likely normal physiological conditions neuroglobin is shown to be present in the ferrous state at approximately 30% of its total cellular concentration. Under hypoxic conditions this rapidly rises to approximately 80%. Temporal analysis of this model indicates that the transition from low concentrations to high concentration of ferrous neuroglobin occurs on the seconds time scale. These findings indicate a potential control model for the anti-apoptotic activity of neuroglobin, under likely physiological conditions, whereby, in normoxic conditions, the anti-apoptotic activity of neuroglobin is maintained at a low level, whilst immediately a transition occurs to a hypoxic situation, as might arise during stroke, the anti-apoptotic activity is drastically increased. In this way the cell avoids unwanted increased oncogenic potential under normal conditions, but the rapid activation of neuroglobin provides anti-apoptotic protection in times of acute hypoxia.

Quantitative ultrasound backscatter for pulsed cavitational ultrasound therapy- histotripsy.

PubMed

Wang, Tzu-yin; Xu, Zhen; Winterroth, Frank; Hall, Timothy L; Fowlkes, J Brian; Rothman, Edward D; Roberts, William W; Cain, Charles A

2009-05-01

Histotripsy is a well-controlled ultrasonic tissue ablation technology that mechanically and progressively fractionates tissue structures using cavitation. The fractionated tissue volume can be monitored with ultrasound imaging because a significant ultrasound backscatter reduction occurs.This paper correlates the ultrasound backscatter reduction with the degree of tissue fractionation characterized by the percentage of remaining normal-appearing cell nuclei on histology.Different degrees of tissue fractionation were generated in vitro in freshly excised porcine kidneys by varying the number of therapeutic ultrasound pulses from 100 to 2000 pulses per treatment location. All ultrasound pulses were 15 cycles at 1 MHz delivered at 100 Hz pulse repetition frequency and 19 MPa peak negative pressure. The results showed that the normalized backscatter intensity decreased exponentially with increasing number of pulses. Correspondingly, the percentage of normal appearing nuclei in the treated area decreased exponentially as well. A linear correlation existed between the normalized backscatter intensity and the percentage of normal appearing cell nuclei in the treated region. This suggests that the normalized backscatter intensity may be a potential quantitative real-time feedback parameter for histotripsy-induced tissue fractionation. This quantitative feedback may allow the prediction of local clinical outcomes, i.e., when a tissue volume has been sufficiently treated.

A novel mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox cycles between peroxynitrite and glutathione.

PubMed

Sun, Chunlong; Du, Wen; Wang, Peng; Wu, Yang; Wang, Baoqin; Wang, Jun; Xie, Wenjun

2017-12-16

Redox homeostasis is important for maintenance of normal physiological functions within cells. Redox state of cells is primarily a consequence of precise balance between levels of reducing equivalents and reactive oxygen species. Redox homeostasis between peroxynitrite (ONOO - ) and glutathione (GSH) is closely associated with physiological and pathological processes, such as prolonged relaxation in vascular tissues and smooth muscle preparations, attenuation of hepatic necrosis, and activation of matrix metalloproteinase-2. We report a two-photon fluorescent probe (TP-Se) based on water-soluble carbazole-based compound, which integrates with organic selenium, to monitor changes in ONOO - /GSH levels in cells. This probe can reversibly respond to ONOO - and GSH and exhibits high selectivity, sensitivity, and mitochondrial targeting. The probe was successfully applied to visualize changes in redox cycles during ONOO - outbreak and antioxidant GSH repair in cells. The probe will lead to significant development on redox events involved in cellular redox regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

PubMed

Giráldez, Servando; Galindo-Moreno, María; Limón-Mortés, M Cristina; Rivas, A Cristina; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2017-07-01

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCF βTrCP /proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G 1 /S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCF βTrCP /proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G 1 -to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G 1 /S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis. © FASEB.

A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

PubMed

Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

2012-07-01

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p < 0.05) in thyroglobulin expression and statistically significant increases (p < 0.05) in Ki-67 and p53 expression as compared with the PTC cohort. A trend toward loss of p16 and p21 expression was noted in the ATC cohort. A trend toward decreased survival was noted with p21 expression. These data indicate disruption of the normal cell cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity.

PubMed

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

2004-11-01

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

N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

PubMed

Mao, Gaowei; Goswami, Monali; Kalen, Amanda L; Goswami, Prabhat C; Sarsour, Ehab H

2016-01-01

The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.

cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior

PubMed Central

Oh, Sangmi; Kawasaki, Ichiro; Park, Jae-Hyung; Shim, Yhong-Hee

2016-01-01

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells. PMID:27770028

cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior.

PubMed

Oh, Sangmi; Kawasaki, Ichiro; Park, Jae-Hyung; Shim, Yhong-Hee

2016-12-07

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells. Copyright © 2016 Oh et al.

Recent progress of Spectrolab high-efficiency space solar cells

NASA Astrophysics Data System (ADS)

Law, Daniel C.; Boisvert, J. C.; Rehder, E. M.; Chiu, P. T.; Mesropian, S.; Woo, R. L.; Liu, X. Q.; Hong, W. D.; Fetzer, C. M.; Singer, S. B.; Bhusari, D. M.; Edmondson, K. M.; Zakaria, A.; Jun, B.; Krut, D. D.; King, R. R.; Sharma, S. K.; Karam, N. H.

2013-09-01

Recent progress in III-V multijunction space solar cell has led to Spectrolab's GaInP/GaAs/Ge triple-junction, XTJ, cells with average 1-sun efficiency of 29% (AM0, 28°C) for cell size ranging from 59 to 72-cm2. High-efficiency inverted metamorphic (IMM) multijunction cells are developed as the next space solar cell architecture. Spectrolab's large-area IMM3J and IMM4J cells have achieved 33% and 34% 1-sun, AM0 efficiencies, respectively. The IMM3J and the IMM4J cells have both demonstrated normalized power retention of 0.86 at 5x1014 e-/cm2 fluence and 0.83 and 0.82 at 1x1015 e-/cm2 fluence post 1-MeV electron radiation, respectively. The IMM cells were further assembled into coverglass-interconnect-cell (CIC) strings and affixed to typical rigid aluminum honeycomb panels for thermal cycling characterization. Preliminary temperature cycling data of two coupons populated with IMM cell strings showed no performance degradation. Spectrolab has also developed semiconductor bonded technology (SBT) where highperformance component subcells were grown on GaAs and InP substrates separately then bonded directly to form the final multijunction cells. Large-area SBT 5-junction cells have achieved a 35.1% efficiency under 1-sun, AM0 condition.

Diagnosis and treatment of diffuse large B-cell lymphoma in an orangutan (Pongo pygmaeus).

PubMed

Ikpatt, Offiong F; Reavill, Drury; Chatfield, Jason; Clubb, Susan; Rosenblatt, Joseph D; Fonte, Glenn; Fan, Yao-Shan; Cray, Carolyn

2014-12-01

Lymphoma is a common malignancy observed in companion animals. This type of naturally occurring neoplasia has been uncommonly reported in great apes. Diffuse large B-cell lymphoma was diagnosed in an 8-yr-old captive orangutan (Pongo pygmaeus) with gastrointestinal disease by histologic and immunohistochemical methodologies. The orangutan was treated with three cycles of combination chemotherapy (intravenous Rituxan, cyclophosphamide, doxorubicin, and vincristine). The primate has been in good health and exhibiting normal behaviors for more than 15 mo following treatment.

Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast Cancer Cell Lines.

DTIC Science & Technology

1999-07-01

medium only, LPS (100 ng/ml), or paclitaxel (35 ^iM), concentrations found to induce maximal levels of mRNA in murine macrophages. Total RNA was...not detected in RNA derived from the DA-3 cells over an 8 h timecourse, even after 40 cycles of PCR amplification, without or with treatment...indicated times after stimulation with LPS or paclitaxel. Isolation of total cellular RNA . For in vitro experiments, culture supematants were removed

Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis.

PubMed

Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

2013-01-01

Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy.

Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

PubMed Central

Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

2013-01-01

Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. PMID:23983455

Preimplantation genetic diagnosis (PGD): the Gothenburg experience.

PubMed

Hanson, C; Jakobsson, A H; Sjögren, A; Lundin, K; Nilsson, L; Wahlström, J; Hardarson, T; Stevic, J; Darnfors, C; Janson, P O; Wikland, M; Hamberger, L

2001-04-01

A program for preimplantation genetic diagnosis of pre-embryos from patients with hereditary disorders was set up in our unit at Sahlgrenska University Hospital in 1994. The majority of the patients were carriers of X-chromosome linked disorders; a few patients were translocation carriers. In this paper we describe our experiences of our first 36 cycles, 30 gender determinations and six analyses of embryos with possible translocations. Conventional hormone replacement treatment with intracytoplasmic sperm injection to fertilize the eggs followed by blastomere biopsy and fluorescent in situ hybridization at the eight cell stage was used for sexing as well as detection of translocations. Out of the 30 cycles in 13 patients for gender determination, blastomere biopsies could be carried out in 25 cycles. Transfer of normal female embryos (XX) was performed in 18 cycles, resulting in five pregnancies (pregnancy rate 27.8%) and an implantation rate of 20% per transfer. Three girls have been born. Hence the take home baby rate was 16.7% per transfer and 10% per started cycle. Six cycles (three patients) for detection of translocations in embryos were performed. Diagnosis was possible in four cycles. Transfer of normal embryos was carried out in one cycle. No pregnancy was achieved. Successful PGD in its clinical application demands close collaboration between a large group of specialists. Even so, the success rate is considerably lower than after conventional IVF or ICSI procedures. Taking into account the stress caused to the parents facing late interruption of pregnancy following conventional prenatal diagnosis we are convinced that this technique is well worthwhile continuing and refining.

Fhit-deficient normal and cancer cells are mitomycin C and UVC resistant

PubMed Central

Ottey, M; Han, S-Y; Druck, T; Barnoski, B L; McCorkell, K A; Croce, C M; Raventos-Suarez, C; Fairchild, C R; Wang, Y; Huebner, K

2004-01-01

To identify functions of the fragile tumour suppressor gene, FHIT, matched pairs of Fhit-negative and -positive human cancer cell clones, and normal cell lines established from Fhit −/− and +/+ mice, were stressed and examined for differences in cell cycle kinetics and survival. A larger fraction of Fhit-negative human cancer cells and murine kidney cells survived treatment with mitomycin C or UVC light compared to matched Fhit-positive cells; ∼10-fold more colonies of Fhit-deficient cells survived high UVC doses in clonigenic assays. The human cancer cells were synchronised in G1, released into S and treated with UVC or mitomycin C. At 18 h post mitomycin C treatment ∼6-fold more Fhit-positive than -negative cells had died, and 18 h post UVC treatment 3.5-fold more Fhit-positive cells were dead. Similar results were obtained for the murine −/− cells. After low UVC doses, the rate of DNA synthesis in −/− cells decreased more rapidly and steeply than in +/+ cells, although the Atr–Chk1 pathway appeared intact in both cell types. UVC surviving Fhit −/− cells appear transformed and exhibit >5-fold increased mutation frequency. This increased mutation burden could explain the susceptibility of Fhit-deficient cells in vivo to malignant transformation. PMID:15494723

Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells.

PubMed

Mishra, Rajesh G; Tseng, Tzu-Ling; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J-F

2016-12-01

Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20μg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction. Copyright © 2016 Elsevier Inc. All rights reserved.

Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

PubMed Central

Rahman, Heshu Sulaiman; Rasedee, Abdullah; Abdul, Ahmad Bustamam; Zeenathul, Nazariah Allaudin; Othman, Hemn Hassan; Yeap, Swee Keong; How, Chee Wun; Hafiza, Wan Abd Ghani Wan Nor

2014-01-01

This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans. PMID:24549090

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

PubMed

Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

2017-01-01

A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

UVA Irradiation Enhances Brusatol-Mediated Inhibition of Melanoma Growth by Downregulation of the Nrf2-Mediated Antioxidant Response

PubMed Central

Wang, Mei; Shi, Guangwei; Bian, Chunxiang; Nisar, Muhammad Farrukh; Guo, Yingying; Wu, Yan; Li, Wei; Huang, Xiao; Jiang, Xuemei; Bartsch, Jörg W.

2018-01-01

Brusatol (BR) is a potent inhibitor of Nrf2, a transcription factor that is highly expressed in cancer tissues and confers chemoresistance. UVA-generated reactive oxygen species (ROS) can damage both normal and cancer cells and may be of potential use in phototherapy. In order to provide an alternative method to treat the aggressive melanoma, we sought to investigate whether low-dose UVA with BR is more effective in eliminating melanoma cells than the respective single treatments. We found that BR combined with UVA led to inhibition of A375 melanoma cell proliferation by cell cycle arrest in the G1 phase and triggers cell apoptosis. Furthermore, inhibition of Nrf2 expression attenuated colony formation and tumor development from A375 cells in heterotopic mouse models. In addition, cotreatment of UVA and BR partially suppressed Nrf2 and its downstream target genes such as HO-1 along with the PI3K/AKT pathway. We propose that cotreatment increased ROS-induced cell cycle arrest and cellular apoptosis and inhibits melanoma growth by regulating the AKT-Nrf2 pathway in A375 cells which offers a possible therapeutic intervention strategy for the treatment of human melanoma. PMID:29670684

Eupatilin, a dietary flavonoid, induces G2/M cell cycle arrest in human endometrial cancer cells.

PubMed

Cho, Jung-Hoon; Lee, Jong-Gyu; Yang, Yeong-In; Kim, Ji-Hyun; Ahn, Ji-Hye; Baek, Nam-In; Lee, Kyung-Tae; Choi, Jung-Hye

2011-08-01

This study is the first to investigate the antiproliferative effect of eupatilin in human endometrial cancer cells. Eupatilin, a naturally occurring flavonoid isolated from Artemisia princeps, has anti-inflammatory, anti-oxidative, and anti-tumor activities. In the present study, we investigated the potential effect of eupatilin on cell growth and its molecular mechanism of action in human endometrial cancer cells. Eupatilin was more potent than cisplatin in inhibiting cell viability in the human endometrial cancer cell lines Hec1A and KLE. Eupatilin showed relatively low cytotoxicity in normal human endometrial cells HES and HESC cells when compared to cisplatin. Eupatilin induced G2/M phase cell cycle arrest in a time- and dose-dependent manner, as indicated by flow cytometry analysis. In addition, treatment of Hec1A cells with eupatilin resulted in a significant increase in the expression of p21(WAF1/CIP1) and in the phosphorylation of Cdc25C and Cdc2. Knockdown of p21 using specific siRNAs significantly compromised eupatilin-induced cell growth inhibition. Interestingly, levels of mutant p53 in Hec1A cells decreased markedly upon treatment with eupatilin, and p53 siRNA significantly increased p21 expression. Moreover, eupatilin modulated the phosphorylation of protein kinases ERK1/2, Akt, ATM, and Chk2. These results suggest that eupatilin inhibits the growth of human endometrial cancer cells via G2/M phase cell cycle arrest through the up-regulation of p21 by the inhibition of mutant p53 and the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway. Copyright © 2011 Elsevier Ltd. All rights reserved.

Overexpression of the Cell Cycle Inhibitor p16INK4a Promotes a Prothrombotic Phenotype Following Vascular Injury in Mice

PubMed Central

Cardenas, Jessica C.; Owens, A. Phillip; Krishnamurthy, Janakiraman; Sharpless, Norman E.; Whinna, Herbert C.; Church, Frank C.

2011-01-01

Objective Age-associated cellular senescence is thought to promote vascular dysfunction. p16INK4a is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16INK4a overexpression on venous thrombosis. Methods and Results Mice overexpressing p16INK4a were studied with four different vascular injury models: (1) ferric chloride (FeCl3) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl3 and vascular ligation to examine thrombus resolution; and (4) LPS administration to initiate inflammation-induced vascular dysfunction. p16INK4a transgenic mice had accelerated occlusion times (13.1 ± 0.4 min) compared to normal controls (19.7 ± 1.1 min) in the FeCl3 model and 12.7 ± 2.0 and 18.6 ± 1.9, respectively in the Rose Bengal model. Moreover, overexpression of p16INK4a delayed thrombus resolution compared to normal controls. In response to LPS treatment, the p16INK4a transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography (CAT) assays. Finally, bone marrow transplantation studies suggested increased p16INK4a expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16INK4a expression in venous thrombosis. Conclusions Venous thrombosis is augmented by overexpression of the cellular senescence gene p16INK4a. PMID:21233453

Blood-gene expression reveals reduced circadian rhythmicity in individuals resistant to sleep deprivation.

PubMed

Arnardottir, Erna S; Nikonova, Elena V; Shockley, Keith R; Podtelezhnikov, Alexei A; Anafi, Ron C; Tanis, Keith Q; Maislin, Greg; Stone, David J; Renger, John J; Winrow, Christopher J; Pack, Allan I

2014-10-01

To address whether changes in gene expression in blood cells with sleep loss are different in individuals resistant and sensitive to sleep deprivation. Blood draws every 4 h during a 3-day study: 24-h normal baseline, 38 h of continuous wakefulness and subsequent recovery sleep, for a total of 19 time-points per subject, with every 2-h psychomotor vigilance task (PVT) assessment when awake. Sleep laboratory. Fourteen subjects who were previously identified as behaviorally resistant (n = 7) or sensitive (n = 7) to sleep deprivation by PVT. Thirty-eight hours of continuous wakefulness. We found 4,481 unique genes with a significant 24-h diurnal rhythm during a normal sleep-wake cycle in blood (false discovery rate [FDR] < 5%). Biological pathways were enriched for biosynthetic processes during sleep. After accounting for circadian effects, two genes (SREBF1 and CPT1A, both involved in lipid metabolism) exhibited small, but significant, linear changes in expression with the duration of sleep deprivation (FDR < 5%). The main change with sleep deprivation was a reduction in the amplitude of the diurnal rhythm of expression of normally cycling probe sets. This reduction was noticeably higher in behaviorally resistant subjects than sensitive subjects, at any given P value. Furthermore, blood cell type enrichment analysis showed that the expression pattern difference between sensitive and resistant subjects is mainly found in cells of myeloid origin, such as monocytes. Individual differences in behavioral effects of sleep deprivation are associated with differences in diurnal amplitude of gene expression for genes that show circadian rhythmicity. © 2014 Associated Professional Sleep Societies, LLC.

p21 controls patterning but not homologous recombination in RPE development.

PubMed

Bishop, A J R; Kosaras, B; Hollander, M C; Fornace, A; Sidman, R L; Schiestl, R H

2006-01-05

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.

Estrous Cyclicity in Mice During Simulated Weightlessness

NASA Technical Reports Server (NTRS)

Moyer, E. L.; Talyansky, Y.; Scott, R. T.; Tash, J. S.; Christenson, L. K.; Alwood, J. S.; Ronca, A. E.

2017-01-01

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n=22), normally loaded HU Cage Pair-Fed controls (CPF; n=22), and Vivarium controls fed ad libitum (VIV; n=10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8+/- 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, and/or due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5% vs 3.0%, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system. Research supported by NNX15AB48G to JST.

DNA damage checkpoint recovery and cancer development

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

Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong, E-mail: lsteng@zju.edu.cn

2015-06-10

Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observedmore » to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint recovery pathway plays a role in overriding tumor barrier in tumorigenesis. • Recovery protein dysregulation and human cancer development is correlated.« less

Intracellular dynamics of cationic and anionic polystyrene nanoparticles without direct interaction with mitotic spindle and chromosomes.

PubMed

Liu, Yuexian; Li, Wei; Lao, Fang; Liu, Ying; Wang, Liming; Bai, Ru; Zhao, Yuliang; Chen, Chunying

2011-11-01

The fate of nanomaterials with different sizes and charges in mitotic cells is of great importance but seldom explored. Herein we investigate the intracellular fate of negatively charged carboxylated polystyrene (COOH-PS) and positively charged amino-modified polystyrene (NH(2)-PS) nanoparticles of three different diameters (50, 100 and 500 nm) on cancer HeLa cells and normal NIH 3T3 cells during the cell cycles. The results showed that all the fluorescent PS nanoparticles differing in size and/or charge did not interact with chromosome reorganization and cytoskeleton assembly during the mitotic process in live cells. They neither disturbed chromosome reorganization nor affected the cytoskeleton reassembly in both normal and cancer cells. However, NH(2)-PS at the size of 50 nm caused G1 phase delay and a decrease of cyclin (D, E) expression, respectively. Moreover, NH(2)-PS displayed higher cellular toxicity and NH(2)-PS of 50 nm disturbed the integrity of cell membranes. Both cationic and anionic PS nanoparticles had a more pronounced effect on normal NIH 3T3 cells than cancer HeLa cell. Our research provides insight into the dynamic fate, intracellular behavior, and the effects of nanoparticles on spindle and chromosomes during cell division, which will enable the optimization of design and selection of much safer nanoparticles for lower risk to human health and widely medical applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Dihydroartemisinin is an inhibitor of ovarian cancer cell growth.

PubMed

Jiao, Yang; Ge, Chun-min; Meng, Qing-hui; Cao, Jian-ping; Tong, Jian; Fan, Sai-jun

2007-07-01

To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.

Qualification testing of general electric 50 A h nickel—cadmium cells with pellon 2536 separator and passivated positive plates

NASA Astrophysics Data System (ADS)

Morrow, George W.

Forty-two, 50 A h nickel—cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a new, nonwoven nylon separator material, Pellon 2536, and the new GE positive plate passivation process. Testing began in May, 1985 at the Naval Weapons Support Center (NWSC) at Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons complete at this writing. After early problems in maintaining test pack temperature control, all packs were performing well but were exhibiting higher than normal charge voltage characteristics.

Qualification testing of General Electric 50 Ah nickel-cadmium cells with Pellon 2536 separator and passivated positive plates

NASA Technical Reports Server (NTRS)

Morrow, George W.

1987-01-01

Forty-two 50 Ah nickel-cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a nonwoven nylon separator material, Pellon 2536, and the GE positive plate nickel attack control gas passivation process. Testing began May, 1985 at the Naval Weapons Support Center (NWSC) in Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons completed. After early problems in maintaining test pack temperature control, all packs are performing well but are exhibiting higher than normal charge voltage characteristics.

Qualification testing of General Electric 50 Ah nickel-cadmium cells with Pellon 2536 separator and passivated positive plates

NASA Astrophysics Data System (ADS)

Morrow, George W.

1987-09-01

Forty-two 50 Ah nickel-cadmium cells were delivered to the Goddard Space Flight Center (GSFC) by General Electric (GE) in February, 1985 for the purpose of evaluating and qualifying a nonwoven nylon separator material, Pellon 2536, and the GE positive plate nickel attack control gas passivation process. Testing began May, 1985 at the Naval Weapons Support Center (NWSC) in Crane, Indiana with GSFC standard initial evaluation tests. Life cycling in both Low Earth Orbit (LEO) and Geosynchronous Orbit (GEO) began in July, 1985 with approximately 6500 LEO cycles and three GEO eclipse seasons completed. After early problems in maintaining test pack temperature control, all packs are performing well but are exhibiting higher than normal charge voltage characteristics.

Novel non-trimethoxylphenyl piperlongumine derivatives selectively kill cancer cells.

PubMed

Zhang, Youjun; Ma, Hao; Wu, Yuelin; Wu, Zhongli; Yao, Zhengguang; Zhang, Wannian; Zhuang, Chunlin; Miao, Zhenyuan

2017-06-01

Piperlongumine (PL) is a natural alkaloid with broad biological activities. Twelve analogues have been designed and synthesized with non-substituted benzyl rings or heterocycles in this work. Most of the compounds showed better anticancer activities than the parent PL without apparent toxicity in normal cells. Elevation of cellular ROS levels was one of the main anticancer mechanisms of these compounds. Cell apoptosis and cell cycle arrest for the best compound ZM90 were evaluated and similar mechanism of action with PL was demonstrated. The SAR was also characterized, providing worthy directions for further optimization of PL compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

PubMed

Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

2007-01-01

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.

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

PubMed Central

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

2014-01-01

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

CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis

PubMed Central

Collins, Carl; Maruthi, N. M.; Jahn, Courtney E.

2015-01-01

A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants. PMID:26022252

Overexpression of ZIC5 promotes proliferation in non-small cell lung cancer

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

Sun, Qi; Shi, Run; Wang, Xin

Background: Non-small cell lung cancer (NSCLC) has become the leading cause of cancer-related deaths. It is therefore urgent that we identify new molecular targets to help cure NSCLC patients. Here, we identified ZIC5 as a potential novel oncogene. Methods: We detected the expression of ZIC5 in tumor and normal tissues of NSCLC patients using quantitative real-time PCR and explored its clinical appearance. We then knocked down ZIC5 to observe changes in NSCLC cell proliferation and metastasis. Nude mouse xenograft models were established to measure ZIC5's function in vivo. Results: Our results revealed that ZIC5 was expressed at dramatically higher levels inmore » NSCLC tumor tissues than in normal tissues. High levels of ZIC5 expression were associated with a higher primary tumor grade. ZIC5 expression was significantly inhibited by small interfering RNA. After silencing ZIC5, the metastatic capacity of NSCLC cells was clearly lower. Knocking down ZIC5 significantly inhibited the proliferation of NSCLC cells, causing the cell cycle to be arrested in G2 phase. Xenograft tumor models showed that knocking down ZIC5 also inhibited tumor growth in vivo. Q-PCR and western blot analysis revealed that ZIC5 expression was closely associated with CCNB1 and CDK1 complex expression, while other cell cycle-related genes showed no significant correlation with ZIC5. Conclusions: Our experiment show that ZIC5 is highly upregulated in NSCLC tumor tissues and suggest that ZIC5 may act as an oncogene by influencing CCNB1 and CDK1 complex expression. ZIC5 may therefore be a potential biomarker and therapeutic target for NSCLC patients.« less

The transcription factor Foxg1 regulates telencephalic progenitor proliferation cell autonomously, in part by controlling Pax6 expression levels

PubMed Central

2011-01-01

Background The transcription factor Foxg1 is an important regulator of telencephalic cell cycles. Its inactivation causes premature lengthening of telencephalic progenitor cell cycles and increased neurogenic divisions, leading to severe hypoplasia of the telencephalon. These proliferation defects could be a secondary consequence of the loss of Foxg1 caused by the abnormal expression of several morphogens (Fibroblast growth factor 8, bone morphogenetic proteins) in the telencephalon of Foxg1 null mutants. Here we investigated whether Foxg1 has a cell autonomous role in the regulation of telencephalic progenitor proliferation. We analysed Foxg1+/+↔Foxg1-/- chimeras, in which mutant telencephalic cells have the potential to interact with, and to have any cell non-autonomous defects rescued by, normal wild-type cells. Results Our analysis showed that the Foxg1-/- cells are under-represented in the chimeric telencephalon and the proportion of them in S-phase is significantly smaller than that of their wild-type neighbours, indicating that their under-representation is caused by a cell autonomous reduction in their proliferation. We then analysed the expression of the cell-cycle regulator Pax6 and found that it is cell-autonomously downregulated in Foxg1-/- dorsal telencephalic cells. We went on to show that the introduction into Foxg1-/- embryos of a transgene designed to reverse Pax6 expression defects resulted in a partial rescue of the telencephalic progenitor proliferation defects. Conclusions We conclude that Foxg1 exerts control over telencephalic progenitor proliferation by cell autonomous mechanisms that include the regulation of Pax6, which itself is known to regulate proliferation cell autonomously in a regional manner. PMID:21418559

An Immunogram for the Cancer-Immunity Cycle: Towards Personalized Immunotherapy of Lung Cancer.

PubMed

Karasaki, Takahiro; Nagayama, Kazuhiro; Kuwano, Hideki; Nitadori, Jun-Ichi; Sato, Masaaki; Anraku, Masaki; Hosoi, Akihiro; Matsushita, Hirokazu; Morishita, Yasuyuki; Kashiwabara, Kosuke; Takazawa, Masaki; Ohara, Osamu; Kakimi, Kazuhiro; Nakajima, Jun

2017-05-01

The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer-immunity cycle by using next-generation sequencing. Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients' human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer-immunity cycle. Three immunogram patterns were observed in patients with lung cancer: T-cell-rich, T-cell-poor, and intermediate. The T-cell-rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid-derived suppressor cells, checkpoint molecules, and immune-inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T-cell-poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T-cell-rich and T-cell-poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. The patient-specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

PubMed

Wang, Ruoxing; Guo, Yan-Lin

2012-10-01

Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. Copyright © 2012 Elsevier Inc. All rights reserved.

Bi-directional signaling: Extracellular Matrix and Integrin Regulation of Breast Tumor Progression

PubMed Central

Gehler, Scott; Ponik, Suzanne M.; Riching, Kristin M; Keely, Patricia J.

2016-01-01

Cell transformation and tumor progression involves a common set of acquired capabilities, including increased proliferation, failure of cell death, self-sufficiency in growth, angiogenesis, and tumor cell invasion and metastasis (1). The stromal environment consists of many cell types, including fibroblasts, macrophages, and endothelial cells, in addition to various extracellular matrix (ECM) proteins that function to support normal tissue maintenance, but have also been implicated in tumor progression (2). Both the chemical and mechanical properties of the ECM have been shown to influence normal and malignant cell behavior. For instance, mesenchymal stem cells differentiate into specific lineages that are dependent on matrix stiffness (3), while tumor cells undergo changes in cell behavior and gene expression in response to matrix stiffness (4). ECM remodeling is implicated in tumor progression and includes changes in both the chemical and mechanical properties of the ECM (5) that can be a result of 1.) increased deposition of stromal ECM, 2.) enhanced contraction of ECM fibrils, and 3.) altered collagen alignment and ECM stiffness. In addition, remodeling of the ECM may alter whether tumor cells employ proteolytic degradation mechanisms during invasion and metastasis. Tumor cells respond to such changes in ECM remodeling through altered intracellular signaling and cell cycle control that lead to enhanced proliferation, loss of normal tissue architecture, and local tumor cell migration and invasion into the surrounding stromal tissue (6). This review will focus on the bi-directional interplay between the mechanical properties of the ECM and changes in integrin-mediated signal transduction events in an effort to elucidate cell behaviors during tumor progression. PMID:23582036

Evaluation program for secondary spacecraft cells: Initial evaluation tests of General Electric Company, 6.0 ampere-hour nickel-cadmium spacecraft cells for the GOES-D, E and F satellite program

NASA Technical Reports Server (NTRS)

Harkness, J. D.

1979-01-01

The tests are to insure that all cells put into the life cycle program are of high quality by the screening of cells found to have electrolyte leakage, internal shorts, low capacity, or inability of any cell to recover its open-circuit voltage above 1.150 volts during the internal short test. Test limits specify those values at which a cell is to be terminated from charge or discharge. Requirements are referenced to normally expected values based on past performance of aerospace nickel-cadmium cells with demonstrated life characteristics. Recommendations for the improvement of the manufacturing processes are presented.

Interactome Analysis of Microtubule-targeting Agents Reveals Cytotoxicity Bases in Normal Cells.

PubMed

Gutiérrez-Escobar, Andrés Julián; Méndez-Callejas, Gina

2017-12-01

Cancer causes millions of deaths annually and microtubule-targeting agents (MTAs) are the most commonly-used anti-cancer drugs. However, the high toxicity of MTAs on normal cells raises great concern. Due to the non-selectivity of MTA targets, we analyzed the interaction network in a non-cancerous human cell. Subnetworks of fourteen MTAs were reconstructed and the merged network was compared against a randomized network to evaluate the functional richness. We found that 71.4% of the MTA interactome nodes are shared, which affects cellular processes such as apoptosis, cell differentiation, cell cycle control, stress response, and regulation of energy metabolism. Additionally, possible secondary targets were identified as client proteins of interphase microtubules. MTAs affect apoptosis signaling pathways by interacting with client proteins of interphase microtubules, suggesting that their primary targets are non-tumor cells. The paclitaxel and doxorubicin networks share essential topological axes, suggesting synergistic effects. This may explain the exacerbated toxicity observed when paclitaxel and doxorubicin are used in combination for cancer treatment. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

p16(INK4a) -mediated suppression of telomerase in normal and malignant human breast cells.

PubMed

Bazarov, Alexey V; Van Sluis, Marjolein; Hines, William C; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-10-01

The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. © 2010 The Authors Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

NASA Technical Reports Server (NTRS)

Lawless, B. DeSales

1999-01-01

A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

Temporal variations in sirtuin expression under normal and ultraviolet B-induced conditions and their correlation to energy levels in normal human epidermal keratinocytes.

PubMed

Pelle, Edward; Dong, Kelly; Pernodet, Nadine

2015-01-01

Sirtuins are post-translational modifiers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identified as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the temporal variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sirtuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. Moreover, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show significant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB-induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1988-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

DNA Damage Response in Cisplatin-Induced Nephrotoxicity

PubMed Central

Zhu, Shiyao; Pabla, Navjotsingh; Tang, Chengyuan; He, Liyu; Dong, Zheng

2015-01-01

Cisplatin and its derivatives are widely used chemotherapeutic drugs for cancer treatment. However, they have debilitating side-effects in normal tissues and induce ototoxicity, neurotoxicity, and nephrotoxicity. In kidneys, cisplatin preferentially accumulates in renal tubular cells causing tubular cell injury and death, resulting in acute kidney injury (AKI). Recent studies have suggested that DNA damage and the associated DNA damage response (DDR) is an important pathogenic mechanism of AKI following cisplatin treatment. Activation of DDR may lead to cell cycle arrest and DNA repair for cell survival or, in the presence of severe injury, kidney cell death. Modulation of DDR may provide novel renoprotective strategies for cancer patients undergoing cisplatin chemotherapy. PMID:26564230

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

PubMed Central

Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

2013-01-01

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

Reduced Mitogenicity of Sera Following Weight Loss in Premenopausal Women

PubMed Central

Azrad, Maria; Chang, Pi-Ling; Gower, Barbara A.; Hunter, Gary R.; Nagy, Tim R.

2011-01-01

We investigated whether serum from normal weight women is less mitogenic and more apoptotic than sera from the same women in the overweight state. Sera from premenopausal women, age (mean ±SEE) 34.6±0.53 years, who were randomized to caloric restriction (CR) (n=13), CR + aerobic exercise (AE) (n=14) or CR + resistance training (RT) (n=20) were used to culture endometrial cancer cells. Phases of the cell cycle were determined, proliferating cell nuclear antigen (PCNA) positivity was used to assess proliferation and apoptosis was assessed by determining cleaved caspase-3 and poly-ADP-ribose polymerase (PARP). Analyses showed that overall, cells grown in sera from the weight-reduced state had significantly more cells in G0/G1 and significantly fewer cells in the S and G2/M phases of the cell cycle than cells grown in sera from the overweight state. PCNA staining confirmed that cells grown in sera from the weight-reduced state had fewer proliferating cells. Cleaved caspase-3 and PARP were not different in cells grown in sera from the weight-reduced state compared to the overweight state. We conclude that weight loss with or without exercise could lower the risk for cancer through changes in serum that result in reduced cellular mitogenicity. PMID:21774593

Mapping of CIP/KIP inhibitors, G1 cyclins D1, D3, E and p53 proteins in the rat term placenta.

PubMed

Korgun, Emin Turkay; Unek, Gozde; Herrera, Emilio; Jones, Carolyn J; Wadsack, Christian; Kipmen-Korgun, Dijle; Desoye, Gernot

2011-09-01

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.

Effects of maple (Acer) plant part extracts on proliferation, apoptosis and cell cycle arrest of human tumorigenic and non-tumorigenic colon cells.

PubMed

González-Sarrías, Antonio; Li, Liya; Seeram, Navindra P

2012-07-01

Phenolic-enriched extracts of maple sap and syrup, obtained from the sugar and red maple species (Acer saccharum Marsh, A. rubrum L., respectively), are reported to show anticancer effects. Despite traditional medicinal uses of various other parts of these plants by Native Americans, they have not been investigated for anticancer activity. Here leaves, stems/twigs, barks and sapwoods of both maple species were evaluated for antiproliferative effects against human colon tumorigenic (HCT-116, HT-29, Caco-2) and non-tumorigenic (CCD-18Co) cells. Extracts were standardized to total phenolic and ginnalin-A (isolated in our laboratory) levels. Overall, the extracts inhibited the growth of the colon cancer more than normal cells (over two-fold), their activities increased with their ginnalin-A levels, with red > sugar maple extracts. The red maple leaf extract, which contained the highest ginnalin-A content, was the most active extract (IC₅₀  = 35 and 16 µg/mL for extract and ginnalin-A, respectively). The extracts were not cytotoxic nor did they induce apoptosis of the colon cancer cells. However, cell cycle analyses revealed that the antiproliferative effects of the extracts were mediated through cell cycle arrest in the S-phase. The results from the current study suggest that these maple plant part extracts may have potential anticolon cancer effects. Copyright © 2011 John Wiley & Sons, Ltd.

Discovery of Potent Antiproliferative Agents Targeting EGFR Tyrosine Kinase Based on the Pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine Scaffold.

PubMed

Aziz, Yasmine Mohamed Abdel; Said, Mohamed Mokhtar; El Shihawy, Hosam Ahmed; Tolba, Mai Fathy; Abouzid, Khaled Abouzid Mohamed

2015-01-01

A series of pyridothieno[3,2-d]pyrimidin-4-amines was designed and synthesized as congeners to the classical 4-anilinoquinazolines as ATP-competitive epidermal growth factor receptor (EGFR) inhibitors. Compound 5a exhibited the most potent and selective inhibitory activity against EGFR with an IC50 value of 36.7 nM. Moreover, compounds 4b and 5a showed remarkable cell growth inhibition against leukemia, central nervous system cancer, and non-small cell lung cancer cell lines that overexpress EGFR, with growth inhibition of 50% (GI50) values of around 10 nM in the full U.S. National Cancer Institute 60 cell panel assay. Cell cycle studies indicated that compounds 4b and 5a induced significant cell cycle arrest in the S-phase and G0/G1, respectively, in addition to boosting P27(kip) expression. Compound 5a did not alter the viability of placental trophoblasts, which reflects its safety for normal cells. The standard COMPARE analyses demonstrated considerable correlation levels between compounds 4b and 5a and erlotinib, with pyridinium chlorochromate (PCC) values of 0.707 and 0.727, respectively.

Mannich bases of 1,2,4-triazole-3-thione containing adamantane moiety: Synthesis, preliminary anticancer evaluation, and molecular modeling studies.

PubMed

Milošev, Milorad Z; Jakovljević, Katarina; Joksović, Milan D; Stanojković, Tatjana; Matić, Ivana Z; Perović, Milka; Tešić, Vesna; Kanazir, Selma; Mladenović, Milan; Rodić, Marko V; Leovac, Vukadin M; Trifunović, Snežana; Marković, Violeta

2017-06-01

A series of 18 novel N-Mannich bases derived from 5-adamantyl-1,2,4-triazole-3-thione was synthesized and characterized using NMR spectroscopy and X-ray diffraction technique. All derivatives were evaluated for their anticancer potential against four human cancer cell lines. Several tested compounds exerted good cytotoxic activities on K562 and HL-60 cell lines, along with pronounced selectivity, showing lower cytotoxicity against normal fibroblasts MRC-5 compared to cancer cells. The effects of compounds 5b, 5e, and 5j on the cell cycle were investigated by flow cytometric analysis. It was found that these compounds cause the accumulation of cells in the subG1 and G1 phases of the cell cycle and induce caspase-dependent apoptosis, while the anti-angiogenic effects of 5b, 5e, and 5j have been confirmed in EA.hy926 cells using a tube formation assay. Further, the interaction of Bax protein with compound 5b was investigated by means of molecular modeling, applying the combined molecular docking/molecular dynamics approach. © 2016 John Wiley & Sons A/S.

MLF1IP promotes normal erythroid proliferation and is involved in the pathogenesis of polycythemia vera.

PubMed

Feng, Gege; Zhang, Tianjiao; Liu, Jinqin; Ma, Xiaotang; Li, Bing; Yang, Lin; Zhang, Yue; Xu, Zefeng; Qin, Tiejun; Zhou, Jiaxi; Huang, Gang; Shi, Lihong; Xiao, Zhijian

2017-03-01

Myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1IP) appears to be an erythroid lineage-specific gene in mice; however, its role in normal erythropoiesis and erythropoietic disorders have not yet been elucidated. Here, we found that MLF1IP is abundantly expressed in human erythroid progenitor cells and that MLF1IP-deficiency reduces cell proliferation resulting from cell cycle arrest. Moreover, MLF1IP expression is exclusively elevated in CFU-E cells from polycythemia vera (PV) patients, and MLF1IP transgenic mice develop a PV-like disorder. Further analyses revealed that the erythroid progenitors and early-stage erythroblasts from these transgenic mice expand by up-regulating cyclin D2 and down-regulating p27 and p21. Thus, our data demonstrate that MLF1IP promotes erythroid proliferation and is involved in the pathogenesis of PV, suggesting that it might be a novel molecular target for erythropoietic disorders. © 2017 Federation of European Biochemical Societies.

CDC25AQ110del: A Novel Cell Division Cycle 25A Isoform Aberrantly Expressed in Non-Small Cell Lung Cancer

PubMed Central

Younis, Rania H.; Cao, Wei; Lin, Ruxian; Xia, Ronghui; Liu, Zhenqiu; Edelman, Martin J.; Mei, Yuping; Mao, Li; Ren, Hening

2012-01-01

Objective Lung cancer remains number one cause of cancer related deaths worldwide. Cell cycle deregulation plays a major role in the pathogenesis of Non-Small Cell Lung Cancer (NSCLC). CDC25A represents a critical cell cycle regulator that enhances cell cycle progression. In this study we aimed to investigate the role of a novel CDC25A transcriptional variant, CDC25AQ110del, on the regulation of the CDC25A protein, and its impact on prognosis of NSCLC patients. Methodology/Principal Findings Here we report a novel CDC25A transcript variant with codon 110 (Glutamine) deletion, that we termed CDC25AQ110del in NSCLC cells. In 9 (75%) of the 12 NSCLC cell lines, CDC25AQ110del expression accounted for more than 20% of the CDC25A transcripts. Biological effects of CDC25AQ110del were investigated in H1299 and HEK-293F cells using UV radiation, flowcytometry, cyclohexamide treatment, and confocal microscopy. Compared to CDC25Awt, CDC25AQ110del protein had longer half-life; cells expressing CDC25AQ110del were more resistant to UV irradiation and showed more mitotic activity. Taqman-PCR was used to quantify CDC25AQ110del expression levels in 88 primary NSCLC tumor/normal tissue pairs. In patients with NSCLC, Kaplan Meier curves showed tumors expressing higher levels of CDC25AQ110del relative to the adjacent lung tissues to have significantly inferior overall survival (P = .0018). Significance Here we identified CDC25AQ110del as a novel transcriptional variant of CDC25A in NSCLC. The sequence-specific nature of the abnormality could be a prognostic indicator in NSCLC patients as well as a candidate target for future therapeutic strategies. PMID:23071577

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

PubMed

Kar, Siddhartha; Wang, Meifang; Ham, Seung Wook; Carr, Brian I

2006-10-01

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

β‑catenin nuclear translocation induced by HIF‑1α overexpression leads to the radioresistance of prostate cancer.

PubMed

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-06-01

Hypoxia-inducible factor‑1α (HIF‑1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF‑1α remain unclear. β‑catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF‑1α and β‑catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4‑2B, were grouped as follows: Negative control (no treatment), HIF‑1α overexpression group (transfected with HIF‑1α overexpression plasmid) and β‑catenin silenced group (transfected with HIF‑1α plasmids and β‑catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4‑2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4‑2B cells, transfection with HIF‑1α overexpression plasmid led to an enhanced β‑catenin nuclear translocation, while β‑catenin silencing inhibited β‑catenin nuclear translocation. The enhanced β‑catenin nuclear translocation induced by HIF‑1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non‑homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF‑1α overexpression enhanced β‑catenin nuclear translocation, which led to the activation of the β‑catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF‑1α overexpression promotes the radioresistance of PCa cells.

Colony-Stimulating Factors for Febrile Neutropenia during Cancer Therapy

PubMed Central

Bennett, Charles L.; Djulbegovic, Benjamin; Norris, LeAnn B.; Armitage, James O.

2014-01-01

A 55-year-old, previously healthy woman received a diagnosis of diffuse large-B-cell lymphoma after the evaluation of an enlarged left axillary lymph node obtained on biopsy. She had been asymptomatic except for the presence of enlarged axillary lymph nodes, which she had found while bathing. She was referred to an oncologist, who performed a staging evaluation. A complete blood count and test results for liver and renal function and serum lactate dehydrogenase were normal. Positron-emission tomography and computed tomography (PET–CT) identified enlarged lymph nodes with abnormal uptake in the left axilla, mediastinum, and retroperitoneum. Results on bone marrow biopsy were normal. The patient’s oncologist recommends treatment with six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone with rituximab (CHOP-R) at 21-day intervals. Is the administration of prophylactic granulocyte colony-stimulating factor (G-CSF) with the first cycle of chemotherapy indicated? PMID:23514290

Compartmentalized Regulation of Parkin-Mediated Mitochondrial Quality Control in the Drosophila Nervous System In Vivo.

PubMed

Sung, Hyun; Tandarich, Lauren C; Nguyen, Kenny; Hollenbeck, Peter J

2016-07-13

In neurons, the normal distribution and selective removal of mitochondria are considered essential for maintaining the functions of the large asymmetric cell and its diverse compartments. Parkin, a E3 ubiquitin ligase associated with familial Parkinson's disease, has been implicated in mitochondrial dynamics and removal in cells including neurons. However, it is not clear how Parkin functions in mitochondrial turnover in vivo, or whether Parkin-dependent events of the mitochondrial life cycle occur in all neuronal compartments. Here, using the live Drosophila nervous system, we investigated the involvement of Parkin in mitochondrial dynamics, distribution, morphology, and removal. Contrary to our expectations, we found that Parkin-deficient animals do not accumulate senescent mitochondria in their motor axons or neuromuscular junctions; instead, they contain far fewer axonal mitochondria, and these displayed normal motility behavior, morphology, and metabolic state. However, the loss of Parkin did produce abnormal tubular and reticular mitochondria restricted to the motor cell bodies. In addition, in contrast to drug-treated, immortalized cells in vitro, mature motor neurons rarely displayed Parkin-dependent mitophagy. These data indicate that the cell body is the focus of Parkin-dependent mitochondrial quality control in neurons, and argue that a selection process allows only healthy mitochondria to pass from cell bodies to axons, perhaps to limit the impact of mitochondrial dysfunction. Parkin has been proposed to police mitochondrial fidelity by binding to dysfunctional mitochondria via PTEN (phosphatase and tensin homolog)-induced putative kinase 1 (PINK1) and targeting them for autophagic degradation. However, it is unknown whether and how the PINK1/Parkin pathway regulates the mitochondrial life cycle in neurons in vivo Using Drosophila motor neurons, we show that parkin disruption generates an abnormal mitochondrial network in cell bodies in vivo and reduces the number of axonal mitochondria without producing any defects in their axonal transport, morphology, or metabolic state. Furthermore, while cultured neurons display Parkin-dependent axonal mitophagy, we find this is vanishingly rare in vivo under normal physiological conditions. Thus, both the spatial distribution and mechanism of mitochondrial quality control in vivo differ substantially from those observed in vitro. Copyright © 2016 the authors 0270-6474/16/367375-17$15.00/0.

Compartmentalized Regulation of Parkin-Mediated Mitochondrial Quality Control in the Drosophila Nervous System In Vivo

PubMed Central

Sung, Hyun; Tandarich, Lauren C.; Nguyen, Kenny

2016-01-01

In neurons, the normal distribution and selective removal of mitochondria are considered essential for maintaining the functions of the large asymmetric cell and its diverse compartments. Parkin, a E3 ubiquitin ligase associated with familial Parkinson's disease, has been implicated in mitochondrial dynamics and removal in cells including neurons. However, it is not clear how Parkin functions in mitochondrial turnover in vivo, or whether Parkin-dependent events of the mitochondrial life cycle occur in all neuronal compartments. Here, using the live Drosophila nervous system, we investigated the involvement of Parkin in mitochondrial dynamics, distribution, morphology, and removal. Contrary to our expectations, we found that Parkin-deficient animals do not accumulate senescent mitochondria in their motor axons or neuromuscular junctions; instead, they contain far fewer axonal mitochondria, and these displayed normal motility behavior, morphology, and metabolic state. However, the loss of Parkin did produce abnormal tubular and reticular mitochondria restricted to the motor cell bodies. In addition, in contrast to drug-treated, immortalized cells in vitro, mature motor neurons rarely displayed Parkin-dependent mitophagy. These data indicate that the cell body is the focus of Parkin-dependent mitochondrial quality control in neurons, and argue that a selection process allows only healthy mitochondria to pass from cell bodies to axons, perhaps to limit the impact of mitochondrial dysfunction. SIGNIFICANCE STATEMENT Parkin has been proposed to police mitochondrial fidelity by binding to dysfunctional mitochondria via PTEN (phosphatase and tensin homolog)-induced putative kinase 1 (PINK1) and targeting them for autophagic degradation. However, it is unknown whether and how the PINK1/Parkin pathway regulates the mitochondrial life cycle in neurons in vivo. Using Drosophila motor neurons, we show that parkin disruption generates an abnormal mitochondrial network in cell bodies in vivo and reduces the number of axonal mitochondria without producing any defects in their axonal transport, morphology, or metabolic state. Furthermore, while cultured neurons display Parkin-dependent axonal mitophagy, we find this is vanishingly rare in vivo under normal physiological conditions. Thus, both the spatial distribution and mechanism of mitochondrial quality control in vivo differ substantially from those observed in vitro. PMID:27413149

Distinct 3' UTRs regulate the life-cycle-specific expression of two TCTP paralogs in Trypanosoma brucei.

PubMed

Jojic, Borka; Amodeo, Simona; Bregy, Irina; Ochsenreiter, Torsten

2018-05-10

The translationally controlled tumor protein (TCTP; also known as TPT1 in mammals) is highly conserved and ubiquitously expressed in eukaryotes. It is involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time, we characterize the expression and function of TCTP in the human and animal pathogen, Trypanosoma brucei We identified two paralogs ( TCTP1 and TCTP2 ) that are differentially expressed in the life cycle of the parasite. The genes have identical 5' untranslated regions (UTRs) and almost identical open-reading frames. The 3'UTRs differ substantially in sequence and length, and are sufficient for the exclusive expression of TCTP1 in procyclic- and TCTP2 in bloodstream-form parasites. Furthermore, we characterize which parts of the 3'UTR are needed for TCTP2 mRNA stability. RNAi experiments demonstrate that TCTP1 and TCTP2 expression is essential for normal cell growth in procyclic- and bloodstream-form parasites, respectively. Depletion of TCTP1 in the procyclic form cells leads to aberrant cell and mitochondrial organelle morphology, as well as enlarged, and a reduced number of, acidocalcisomes. © 2018. Published by The Company of Biologists Ltd.

Comparative vaginal cytology of the estrous cycle of black-footed ferrets (Mustela nigripes), Siberian polecats (M. eversmanni), and domestic ferrets (M. putorius furo).

PubMed

Williams, E S; Thorne, E T; Kwiatkowski, D R; Lutz, K; Anderson, S L

1992-01-01

Vaginal cytology and vulva size were used to characterize the reproductive cycle of female black-footed ferrets (Mustela nigripes), Siberian polecats (M. eversmanni), and domestic ferrets (M. putorius furo). Emphasis was on black-footed ferrets because of the need to breed these critically endangered animals and on Siberian polecats because of the close taxonomic relationship to black-footed ferrets. Vaginal cytology of the 3 species of ferret is similar. Proestrus was characterized by an increasing percentage of superficial epithelial cells and enlargement of the vulva. During estrus, superficial cells were usually greater than or equal to 90% of epithelial cells in the vaginal lavage and after several days were fully keratinized. Neutrophils were more common during all stages of the estrous cycle in domestic ferrets than they were in the other species. Following copulation, percentage of superficial calls in the vagina declined and vulva swelling subsided. Large cells, probably of uterine symplasma origin, were observed in vaginal lavages following whelping or pseudopregnancy. Vaginal cytology is extremely useful in the reproductive management of black-footed ferrets and Siberian polecats. Knowledge of normal vaginal cytology could be applied to the diagnosis of female reproductive abnormalities in all 3 species.

Premature aging/senescence in cancer cells facing therapy: good or bad?

PubMed

Gonzalez, Llilians Calvo; Ghadaouia, Sabrina; Martinez, Aurélie; Rodier, Francis

2016-02-01

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent "proliferative arrest" commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.

Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

PubMed Central

Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

2016-01-01

The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

Aloe vera inhibits proliferation of human breast and cervical cancer cells and acts synergistically with cisplatin.

PubMed

Hussain, Arif; Sharma, Chhavi; Khan, Saniyah; Shah, Kruti; Haque, Shafiul

2015-01-01

Many of the anti-cancer agents currently used have an origin in natural sources including plants. Aloe vera is one such plant being studied extensively for its diverse health benefits, including cancer prevention. In this study, the cytotoxic potential of Aloe vera crude extract (ACE) alone or in combination with cisplatin in human breast (MCF-7) and cervical (HeLa) cancer cells was studied by cell viability assay, nuclear morphological examination and cell cycle analysis. Effects were correlated with modulation of expression of genes involved in cell cycle regulation, apoptosis and drug metabolism by RT-PCR. Exposure of cells to ACE resulted in considerable loss of cell viability in a dose- and time-dependent fashion, which was found to be mediated by through the apoptotic pathway as evidenced by changes in the nuclear morphology and the distribution of cells in the different phases of the cell cycle. Interestingly, ACE did not have any significant cytotoxicity towards normal cells, thus placing it in the category of safe chemopreventive agent. Further, the effects were correlated with the downregulation of cyclin D1, CYP 1A1, CYP 1A2 and increased expression of bax and p21 in MCF-7 and HeLa cells. In addition, low dose combination of ACE and cisplatin showed a combination index less than 1, indicating synergistic growth inhibition compared to the agents applied individually. In conclusion, these results signify that Aloe vera may be an effective anti-neoplastic agent to inhibit cancer cell growth and increase the therapeutic efficacy of conventional drugs like cispolatin. Thus promoting the development of plant-derived therapeutic agents appears warranted for novel cancer treatment strategies.

Identification of Key Transcription Factors Associated with Lung Squamous Cell Carcinoma

PubMed Central

Zhang, Feng; Chen, Xia; Wei, Ke; Liu, Daoming; Xu, Xiaodong; Zhang, Xing; Shi, Hong

2017-01-01

Background Lung squamous cell carcinoma (lung SCC) is a common type of lung cancer, but its mechanism of pathogenesis is unclear. The aim of this study was to identify key transcription factors in lung SCC and elucidate its mechanism. Material/Methods Six published microarray datasets of lung SCC were downloaded from Gene Expression Omnibus (GEO) for integrated bioinformatics analysis. Significance analysis of microarrays was used to identify differentially expressed genes (DEGs) between lung SCC and normal controls. The biological functions and signaling pathways of DEGs were mapped in the Gene Otology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, respectively. A transcription factor gene regulatory network was used to obtain insights into the functions of DEGs. Results A total of 1,011 genes, including 539 upregulated genes and 462 downregulated genes, were filtered as DEGs between lung SCC and normal controls. DEGs were significantly enriched in cell cycle, DNA replication, p53 signaling pathway, pathways in cancer, adherens junction, and cell adhesion molecules signaling pathways. There were 57 transcription factors identified, which were used to construct a regulatory network. The network consisted of 736 interactions between 49 transcription factors and 486 DEGs. NFIC, BRCA1, and NFATC2 were the top 3 transcription factors that had the highest connectivity with DEGs and that regulated 83, 82, and 75 DEGs in the network, respectively. Conclusions NFIC, BRCA1, and NFATC2 might be the key transcription factors in the development of lung SCC by regulating the genes involved in cell cycle and DNA replication pathways. PMID:28081052

Synthesis and biological evaluation of 3-substituted-4-(4-methylthio phenyl)-1H-pyrrole derivatives as potential anticancer agents.

PubMed

Lan, Lan; Qin, Weixi; Zhan, Xiaoping; Liu, Zenglu; Mao, Zhenmin

2014-01-01

A novel series of 3-substituted-4-(4-methylthio phenyl)-1H-pyrrole derivatives were synthesized via Van Leusen pyrrole synthesis. The in vitro anticancer activity against a panel of 16 cancer cell lines and 2 normal cell lines was investigated by MTT assay. It was found that some of the pyrrole compounds showed similar antiproliferative activity against cancer cells compared with Paclitaxel, but little impact on normal cell lines, which indicated that the novel pyrrole derivatives could be used as potential anticancer candidates for possessing both selectivity and good therapeutic efficacy. Structure-activity relationship analysis found that 3-phenylacetyl-4- (4-methylthio phenyl)-1H-pyrrole derivatives displayed the most strong anticancer activity, among which [4-(4-methylthio phenyl)-1H-pyrrol- 3-yl] (4-methoxy phenyl) methanone (3j) was employed to investigate the effect of these pyrrole analogues on cell cycle by propidium iodide (PI) staining on cell flow cytometry. Cell necrotic effect of 10.0 µM 3j against MGC80-3 cells were also observed under fluorescence microscope and transmission electron microscope by ultrathin sections observation.

Antiproliferative Activity of Egg Yolk Peptides in Human Colon Cancer Cells.

PubMed

Yousr, Marwa N; Aloqbi, Akram A; Omar, Ulfat M; Howell, Nazlin K

2017-01-01

Egg yolk peptides were successfully prepared from egg yolk protein by-products after lecithin extraction. Defatted egg yolk protein was hydrolyzed with pepsin and pancreatin and purified by gel filtration to produce egg yolk gel filtration fraction (EYGF-33) with antiproliferative activity. The highlight of this study was that the peptide EYGF-33 (1.0 mg/ml) significantly inhibits cell viability of colon cancer cells (Caco-2) with no inhibitory effects on the viability of human colon epithelial normal cells (HCEC) after 48 h. Reduced cell viability can be explained by cell cycle arrest in the S-phase in which DNA replication normally takes place. EYGF-33 significantly enhanced the production of superoxide anions in the mitochondria of Caco-2 cells; this could activate a mitochondrial apoptotic pathway leading to typical Poly Adenosine diphosphate-ribose polymerase (PARP) cleavage as observed in the Western blot result. The induction of apoptotic cell death by EYGF-33 was supported by the externalization of phosphatidylserine (PS). However, further elucidation of the mechanism of EYGF-33-mediated apoptosis would provide further support for its use as a potential therapeutic and chemopreventive agent.

An authentic imaging probe to track cell fate from beginning to end.

PubMed

Lee, Seung Koo; Mortensen, Luke J; Lin, Charles P; Tung, Ching-Hsuan

2014-10-17

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Initial evaluation tests of General Electric Company 26.5 ampere-hour nickel-cadmium spacecraft cells with auxiliary electrodes for the TIROS-N and NOAA-A satellites

NASA Technical Reports Server (NTRS)

Harkness, J. D.

1978-01-01

This evaluation test program had the purpose to insure that all cells put into the life cycle program are of high quality by the screening of cells found to have electrolyte leakage, internal shorts, low capacity, or inability of any cell to recover its open-circuit voltage above 1.150 volts during the internal short test. Test limits specify those values at which a cell is to be terminated from charge or discharge. Requirements are referenced to as normally expected values based on past performance of aerospace nickel-cadmium cells with demonstrated life characteristics. A requirement does not constitute a limit for discontinuance from test.

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth.

PubMed

Qi, Ruhu; John, Peter Crook Lloyd

2007-07-01

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.

Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

PubMed

Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla

2016-10-01

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

PubMed Central

Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid

2016-01-01

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. PMID:27402865

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Identification of 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate as a novel inhibitor to Y box binding protein-1 (YB-1) and its therapeutic actions against breast cancer.

PubMed

Gunasekaran, Vinoth Prasanna; Nishi, Kumari; Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu; Mathan, Ganeshan

2018-04-30

In spite of advances in breast cancer treatment and early diagnosis, drug toxicity, cancer relapse, multidrug resistance and metastasis are the major impediment to the developments of efficient drugs. However, unique druggable targets of cancer cells distinct from the normal cells provide new rationale in cancer treatment. Previous reports clearly emphasize the differential expression and localization of Y box binding protein-1 (YB-1) between normal breast tissues and different stages of breast cancer. Y box binding protein-1 is DNA as well as RNA binding protein involved in transcription and translation regulation of various proteins involved in cancer progression, apoptosis, cell cycle, epithelial to mesenchymal transition (EMT) and drug resistance. Particularly, during doxorubicin (DOX) treatment and cancer relapse conditions, YB-1 expression was very high in breast cancer tissues and localized in to nucleus which further favours DOX efflux and metastasis. Moreover, siRNA mediated silencing of YB-1 reduces breast cancer progression and metastasis. In this rationale, using an array of computational methods, 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate (DPI) has been screened out as a drug-likeness antagonist to the YB-1for cancer treatment. In this study, we determined that DPI was toxic to breast cancer cell lines as individual drug as well as in combination with DOX. Moreover, immunofluorescence and confocal studies showed that DPI decreases DOX induced YB-1 nuclear translocation and increases DOX accumulation in breast cancer cell line. A G1/G0 phase cell cycle arrest and apoptosis was also induced by DPI. Moreover, DPI modulated YB-1 downstream targets such as p53, caspase-3, CDK-1 which are involved in cell cycle progression and apoptosis. Further, metastatic functional analysis revealed that DPI inhibits cell adhesion, migration, invasion in aggressive metastatic cell line and inhibits angiogenesis in chick embryonic chorioallantoic membrane (CAM) model. Meanwhile, DPI alters the expression of YB-1 downstream targets which are involved in metastasis such as VEGFR, caveolin, E-cadherin, cytokeratins, desmin and vimentin in MDA-MB-231 xenograft in chick embryonic CAM membrane. The results clearly demonstrated that DPI inhibited YB-1 nuclear translocation, thereby exhibited anti-apoptotic, anti-proliferative and anti-metastatic activities and increases the therapeutic potential of commercial breast cancer drug doxorubicin. Copyright © 2017 Elsevier B.V. All rights reserved.

Myeloperoxidase in blood neutrophils during normal and abnormal menstrual cycles in women of reproductive age.

PubMed

Shibata, T; Sakamoto, J; Osaka, Y; Neyatani, N; Fujita, S; Oka, Y; Takagi, H; Mori, H; Fujita, H; Tanaka, Y; Sasagawa, T

2017-04-01

We previously reported that granulocyte colony-stimulating factor (G-CSF) plays a critical role in ovulation, suggesting that neutrophils may maintain ovulation. We assessed myeloperoxidase (MPO), a major and specific enzyme of neutrophils, in women with abnormal and normal menstrual cycles to clarify the relationship between MPO and ovulation. We analyzed MPO activity in blood neutrophils of women with abnormal menstrual cycles (indicative of anovulation, n = 12) and age- and body mass index-matched normal menstrual cycles (indicative of ovulation, n = 24) using two parameters as a marker of MPO, Neut X and mean peroxidase index (MPXI). MPO of women with abnormal menstrual cycles was significantly lower than that of women with normal menstrual cycles [Neut X: 62.6 ± 1.1 (mean ± standard error of the mean) vs. 66.2 ± 0.3, P = 0.009; MPXI: -0.54 ± 1.66 vs. 4.91 ± 0.53, P = 0.008]. Among women with normal menstrual cycles, MPO was highest in the follicular phase (Neut X: 67.0 ± 0.3; P = 0.033). The difference in MPO between women with abnormal and normal menstrual cycles and the upregulation of MPO before ovulation suggest that neutrophils and MPO are closely related to ovulation. © 2016 John Wiley & Sons Ltd.

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth.

PubMed

Li, H; Li, J; Su, Y; Fan, Y; Guo, X; Li, L; Su, X; Rong, R; Ying, J; Mo, X; Liu, K; Zhang, Z; Yang, F; Jiang, G; Wang, J; Zhang, Y; Ma, D; Tao, Q; Han, W

2014-06-12

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

Sonoporation as a cellular stress: induction of morphological repression and developmental delays.

PubMed

Chen, Xian; Wan, Jennifer M F; Yu, Alfred C H

2013-06-01

For sonoporation to be established as a drug/gene delivery paradigm, it is essential to account for the biological impact of this membrane permeation strategy on living cells. Here we provide new insight into the cellular impact of sonoporation by demonstrating in vitro that this way of permeating the plasma membrane may inadvertently induce repressive cellular features even while enhancing exogenous molecule uptake. Both suspension-type (HL-60) and monolayer (ZR-75-30) cells were considered in this investigation, and they were routinely exposed to 1-MHz pulsed ultrasound (pulse length, 100 cycles; pulse repetition frequency, 1 kHz; exposure period, 60 s) with calibrated field profile (spatial-averaged peak negative pressure, 0.45 MPa) and in the presence of microbubbles (cell:bubble ratio, 10:1). The post-exposure morphology of sonoporated cells (identified as those with calcein internalization) was examined using confocal microscopy, and their cell cycle progression kinetics were analyzed using flow cytometry. Results show that for both cell types investigated, sonoporated cells exhibited membrane shrinkage and intra-cellular lipid accumulation over a 2-h period. Also, as compared with normal cells, the deoxyribonucleic acid synthesis duration of sonoporated cells was significantly lengthened, indicative of a delay in cell cycle progression. These features are known to be characteristics of a cellular stress response, suggesting that sonoporation indeed constitutes as a stress to living cells. This issue may need to be addressed in optimizing sonoporation for drug/gene delivery purposes. On the other hand, it raises opportunities for developing other therapeutic applications via sonoporation. Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Determination of the cellular retinoic-acid-binding protein in dysplastic epithelia of the cervix uteri, differentiated into apo and holo forms.

PubMed

Volz-Köster, S; Volz, J; Kiefer, A; Biesalski, H K

2000-01-01

The appearance of the cervical mucosa is regulated by different factors including retinoic acid. Hormone-dependent alteration of the cervix uteri mucosa is accompanied by a decrease or increase of cytoplasmatic retinoic-acid-binding protein (CRABP). To elucidate whether this hormone-dependent alteration of CRABP is preserved in the case of neoplasms of the cervix uteri, we measured the level of total and apo-CRABP in normal and neoplastically transformed cervical cells. In a prospective pilot study, standardised biopsies of normal epithelium and cervical intra-epithelial neoplasm grade 3 (CIN III) were taken from 24 patients. A newly developed method was used to determine the intra-epithelial level of apo- and total CRABP. The concentration of total CRABP in normal squamous epithelium compared with that in intra-epithelial neoplasm grade 3 is very significantly lower in the CIN III areas (normal: 3.66 +/- 1.46 pmol/ mg wet weight +/- SD; CIN III 1.43 +/- 0.59 pmol/mg P < 0.01). In addition CRABP in the apo form is lower in normal than in neoplastic epithelium (Wilcoxon test for paired non-parametric values: P < 0.05; mean for all patients: normal: 1.65 + 0.82 pmol/mg; CIN III: 1.14 +/- 0.23 pmol/mg). From our results we conclude that, in neoplastically transformed cells, the hormone-dependent CRABP cycle is interrupted. Whether this has consequences for the further development of the neoplastic cells has to be elucidated.

Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis

PubMed Central

Srivastava, Shikha; Somasagara, Ranganatha R.; Hegde, Mahesh; Nishana, Mayilaadumveettil; Tadi, Satish Kumar; Srivastava, Mrinal; Choudhary, Bibha; Raghavan, Sathees C.

2016-01-01

Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy. PMID:27068577

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

PubMed Central

Domínguez-Calderón, Alaide; Ávila-Flores, Antonia; Ponce, Arturo; López-Bayghen, Esther; Calderón-Salinas, José-Víctor; Luis Reyes, José; Chávez-Munguía, Bibiana; Segovia, José; Angulo, Carla; Ramírez, Leticia; Gallego-Gutiérrez, Helios; Alarcón, Lourdes; Martín-Tapia, Dolores; Bautista-García, Pablo; González-Mariscal, Lorenza

2016-01-01

Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size. PMID:27009203