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Sample records for s-phase-dependent cell cycle

  1. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R.

    Science.gov (United States)

    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.

  2. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

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    Liang Dong

    2010-07-01

    Full Text Available Abstract Background 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. Results 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. Conclusions 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.

  3. Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

    International Nuclear Information System (INIS)

    Bhadriraju, Kiran; Hansen, Linda K.

    2004-01-01

    Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation

  4. Angular-dependent light scattering from cancer cells in different phases of the cell cycle.

    Science.gov (United States)

    Lin, Xiaogang; Wan, Nan; Weng, Lingdong; Zhou, Yong

    2017-10-10

    Cancer cells in different phases of the cell cycle result in significant differences in light scattering properties. In order to harvest cancer cells in particular phases of the cell cycle, we cultured cancer cells through the process of synchronization. Flow cytometric analysis was applied to check the results of cell synchronization and prepare for light scattering measurements. Angular-dependent light scattering measurements of cancer cells arrested in the G1, S, and G2 phases have been performed. Based on integral calculations for scattering intensities from 5° to 10° and from 110° to 150°, conclusions have been reached. Clearly, the sizes of the cancer cells in different phases of the cell cycle dominated the forward scatter. Accompanying the increase of cell size with the progression of the cell cycle, the forward scattering intensity also increased. Meanwhile, the DNA content of cancer cells in every phase of the cell cycle is responsible for light scattering at large scatter angles. The higher the DNA content of cancer cells was, the greater the positive effect on the high-scattering intensity. As expected, understanding the relationships between the light scattering from cancer cells and cell cycles will aid in the development of cancer diagnoses. Also, it may assist in the guidance of antineoplastic drugs clinically.

  5. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    Science.gov (United States)

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  6. Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells

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    Xiaotong Li

    2018-03-01

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

  7. Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

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    Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

    1984-11-01

    During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

  8. Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4).

    Science.gov (United States)

    Shibui, S; Hoshino, T; Iwasaki, K; Nomura, K; Jastreboff, M M

    1989-05-01

    A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.

  9. Cell-cycle phase specificity of chloroethylnitrosoureas

    International Nuclear Information System (INIS)

    Linfoot, P.A.

    1986-01-01

    Although the cancer chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is considered a non-cell cycle phase specific drug, it has been shown to produce differential cell killing in G 1 , S, and G 2 /M phase cells, with S phase cells appearing relatively resistant. Studies of cell cycle phase specific cell killing produced by nitrosoureas with different chemical reactivities, clearly indicated that the ability of compounds to cross-link DNA was important in determining their phase specificity. Cells that lacked guanine O 6 -alkytransferase activity showed similar patterns of BCNU phase specificity regardless of their intrinsic sensitivity to BCNU. DNA inter-strand cross-linking, as measured by alkaline elution, was similar in cells exposed to BCNU in G 1 or S phase. 3 H [1-chloroethyl-1nitrosourea] binding to DNA was the same in G 1 , S and G 2 /M phase cells indicating that phase-specific differences in drug uptake and intracellular drug dose were not responsible for phase specific cell kill. These studies suggest that cross-link lesions, other than DNA inter-strand cross-links, and/or effects on DNA repair, other than guanine O 6 -alkyltransferase, are additional important determinants of BCNU phase specific cell killing

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

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

    2018-05-22

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

  11. S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

    Science.gov (United States)

    Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

    2018-05-04

    Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

  12. S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Alexandersen, Søren

    1997-01-01

    We examined replication of the autonomous parovirus Aleutian mink disease parovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cyc...

  13. Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

    Science.gov (United States)

    Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

    2018-06-01

    Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

  14. Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide

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    Zhang Samuel S

    2010-12-01

    Full Text Available Abstract Background Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported. Methods C6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP. Results Treatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation. Conclusion Our results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle

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

    Science.gov (United States)

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

    2017-03-01

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

  16. Rapid cell cycle analysis by measurement of the radioactivity per cell in a narrow window in S phase (RCSsub(i))

    International Nuclear Information System (INIS)

    Gray, J.W.; Carver, J.H.; George, Y.S.; Mendelsohn, M.L.

    1977-01-01

    A new rapid method for the cell cycle analysis of asynchronously growing cells is presented. The new method is an alternative to the more time consuming and subjective fraction of labeled mitoses (FLM) method. Like the FLM method, all cells in the S phase of the cell cycle are marked by pulse labeling with a radioactive DNA precursor. The subsequent progress of the cohort of cells thus labeled is monitored through a narrow window in the cell cycle. The window is defined by a narrow range of DNA contents corresponding to cells in mid-S phase and is designated Ssub(i). The cellular DNA content is measured by flow cytometry and the cells in the window Ssub(i) are selected by electronic cell sorting. The radioactivity per cell in Ssub(i) (RCSsub(i)) is determined by liquid scintillation counting. The duration of S phase and of the total cycle and the dispersions therein are determined from the oscillation of the RCSsub(i) values with time. The complete cell cycle analysis can be accomplished in as little as 1 day following the collection of samples. Exponentially growing Chinese hamster ovary (CHO) cells were analyzed according to the RCSsub(i) method and the FLM method. It is demonstrated that the two techniques give essentially the same results. (author)

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

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

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

  18. Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression.

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    Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian; Kohlwein, Sepp D

    2015-03-10

    Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.

  19. Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair

    International Nuclear Information System (INIS)

    Rowley, R.

    1985-01-01

    Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G 2 . The sensitivity of Chinese hamster ovary cells to G 2 arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G 2 . This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G 2 arrest and/or by changes in capability for postirradiation recovery from G 2 arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G 2 arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G 2 arrest, while inhibiting repair of G 2 arrest-causing damage makes such an analysis possible. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G 2 arrest was expressed. The duration of G 2 arrest was independent of the length of the prior caffeine exposure. This finding indicates that the target for G 2 arrest induction is present throughout the cell cycle and that the level of G 2 arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G 2 arrest

  20. Segmentation and classification of cell cycle phases in fluorescence imaging.

    Science.gov (United States)

    Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

    2009-01-01

    Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

  1. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

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    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

  2. Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells.

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    Bruni, R; Roizman, B

    1998-11-01

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

  3. Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

    Science.gov (United States)

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

    2011-01-01

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

  4. Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

    International Nuclear Information System (INIS)

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

    1988-01-01

    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 an 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

  5. Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1

    DEFF Research Database (Denmark)

    Walmod, Peter S.; Hartmann-Petersen, Rasmus; Prag, S.

    2004-01-01

    comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner...... for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility...... was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels...

  6. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-09-15

    When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

  7. Impact of cycling cells and cell cycle regulation on Hydra regeneration.

    Science.gov (United States)

    Buzgariu, Wanda; Wenger, Yvan; Tcaciuc, Nina; Catunda-Lemos, Ana-Paula; Galliot, Brigitte

    2018-01-15

    Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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 (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.

  9. Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

    Science.gov (United States)

    Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

    2011-01-01

    tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

    Science.gov (United States)

    Itoh, Tomohiro; Ono, Akito; Kawaguchi, Kaori; Teraoka, Sayaka; Harada, Mayo; Sumi, Keitaro; Ando, Masashi; Tsukamasa, Yasuyuki; Ninomiya, Masayuki; Koketsu, Mamoru; Hashizume, Toshiharu

    2018-05-01

    The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Wenmeng Wang

    Full Text Available High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC and human aortic endothelial cells (HAEC down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin.

  12. Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

    Science.gov (United States)

    Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

    2016-07-01

    Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  13. Cell Cycle Deregulation in the Neurons of Alzheimer’s Disease

    Science.gov (United States)

    Moh, Calvin; Kubiak, Jacek Z.; Bajic, Vladan P.; Zhu, Xiongwei; Smith, Mark A.

    2018-01-01

    The cell cycle consists of four main phases: G1, S, G2, and M. Most cells undergo these cycles up to 40–60 times in their life. However, neurons remain in a nondividing, nonreplicating phase, G0. Neurons initiate but do not complete cell division, eventually entering apoptosis. Research has suggested that like cancer, Alzheimer’s disease (AD) involves dysfunction in neuronal cell cycle reentry, leading to the development of the two-hit hypothesis of AD. The first hit is abnormal cell cycle reentry, which typically results in neuronal apoptosis and prevention of AD. However, with the second hit of chronic oxidative damage preventing apoptosis, neurons gain “immortality” analogous to tumor cells. Once both of these hits are activated, AD can develop and produce senile plaques and neurofibrillary tangles throughout brain tissue. In this review, we propose a mechanism for neuronal cell cycle reentry and the development of AD. PMID:21630160

  14. Backup pathways of NHEJ in cells of higher eukaryotes: Cell cycle dependence

    International Nuclear Information System (INIS)

    Iliakis, George

    2009-01-01

    DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs, DNA ligase IV, XRCC4 and XLF/Cernunnos (D-NHEJ) as central components. Work carried out in our laboratory and elsewhere shows that when this pathway is chemically or genetically compromised, cells do not shunt DSBs to homologous recombination repair (HRR) but instead use another form of NHEJ operating as a backup (B-NHEJ). Here I review our efforts to characterize this repair pathway and discuss its dependence on the cell cycle as well as on the growth conditions. I present evidence that B-NHEJ utilizes ligase III, PARP-1 and histone H1. When B-NHEJ is examined throughout the cell cycle, significantly higher activity is observed in G2 phase that cannot be attributed to HRR. Furthermore, the activity of B-NHEJ is compromised when cells enter the plateau phase of growth. Together, these observations uncover a repair pathway with unexpected biochemical constitution and interesting cell cycle and growth factor regulation. They generate a framework for investigating the mechanistic basis of HRR contribution to DSB repair.

  15. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

    Science.gov (United States)

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

    2004-05-01

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

  16. Cell cycle disturbances in slowly growing sublines isolated from X-irradiated L5178Y-S cell populations

    International Nuclear Information System (INIS)

    Beer, J.Z.; Bocian, E.; Budzicka, E.; Szumiel, I.; Ziemba-Zak, B.; Kopec, M.

    1974-01-01

    Cell cycle was analyzed autoradiographically in a test line of murine leukaemic lymphoblasts L5178Y-S and in two slowly growing sublines isolated from cell cultures irradiated with 300 rad of X-rays. It was found that prolongation of the cell cycle in the slowly growing sublines is connected primarily with delayed progression through G2 phase. This conclusion was further supported by results of determination of DNA content per cell in 13 slowly growing cell sublines and karyotype analysis of 18 sublines. No correlation was found between a sublines' mean doubling time and its chromosome number whereas DNA content per cell was clearly dependent on the growth rate. (author)

  17. A signature-based method for indexing cell cycle phase distribution from microarray profiles

    Directory of Open Access Journals (Sweden)

    Mizuno Hideaki

    2009-03-01

    Full Text Available Abstract Background The cell cycle machinery interprets oncogenic signals and reflects the biology of cancers. To date, various methods for cell cycle phase estimation such as mitotic index, S phase fraction, and immunohistochemistry have provided valuable information on cancers (e.g. proliferation rate. However, those methods rely on one or few measurements and the scope of the information is limited. There is a need for more systematic cell cycle analysis methods. Results We developed a signature-based method for indexing cell cycle phase distribution from microarray profiles under consideration of cycling and non-cycling cells. A cell cycle signature masterset, composed of genes which express preferentially in cycling cells and in a cell cycle-regulated manner, was created to index the proportion of cycling cells in the sample. Cell cycle signature subsets, composed of genes whose expressions peak at specific stages of the cell cycle, were also created to index the proportion of cells in the corresponding stages. The method was validated using cell cycle datasets and quiescence-induced cell datasets. Analyses of a mouse tumor model dataset and human breast cancer datasets revealed variations in the proportion of cycling cells. When the influence of non-cycling cells was taken into account, "buried" cell cycle phase distributions were depicted that were oncogenic-event specific in the mouse tumor model dataset and were associated with patients' prognosis in the human breast cancer datasets. Conclusion The signature-based cell cycle analysis method presented in this report, would potentially be of value for cancer characterization and diagnostics.

  18. Cell-cycle-dependent drug-resistant quiescent cancer cells induce tumor angiogenesis after chemotherapy as visualized by real-time FUCCI imaging

    Science.gov (United States)

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

    2017-01-01

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

  19. DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

    Science.gov (United States)

    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

  20. DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

    Directory of Open Access Journals (Sweden)

    Monika Hlavová

    Full Text Available 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.

  1. Dose-rate effects in plateau-phase cultures of S3 HeLa and V79 cells

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Bedford, J.S.; Bailey, S.M.

    1979-01-01

    Dose-rate effects on cell survival were studied for log-, fed plateau-, and unfed plateau-phase cultures of V79 and S3 HeLa cells. For log-phase cultures, repair, cell-cycle redistribution, and cell division during exposure can contribute to the overall dose-rate effect, but their relative contributions are difficult to determine. With plateau-phase cultures, the cell-cycle times are greatly lengthened, for those cells that are in cycle. Hence, the contribution to the overall dose-rate effect of cell-cycle redistribution and cell division during the exposure could be minimized using plateau-phase cultures. With respect to the acute dose-survival curves, there was a clear loss in effectiveness when the dose rate was lowered to 154 rad/hr for both fed and unfed plateau-phase HeLa and V79 cells. There was no further reduction in effectiveness per unit dose, however, when the dose rate was reduced to 55 rad/hr. Since there was virtually no cell division or cell-cycle redistribution, it may be that a limit to the repair-dependent dose-rate effect at 37 0 C has been reached at a dose rate of 154 rad/hr

  2. Cell cycle phase of nondividing cells in aging human cell cultures determined by DNA content and chromosomal constitution

    International Nuclear Information System (INIS)

    Yanishevsky, R.M.

    1975-01-01

    Human diploid cell cultures, strain WI-38, have a finite proliferative capacity and have been proposed as a model of biological aging. To identify the cell cycle phase of the nondividing cells, cultures of various ages were exposed to 3 Hdt for 48 hours to label dividing cells, then the cycle phase was identified for individual cells by one of two methods, and finally, the proliferative status of the same cells was scored by autoradiographic evidence of 3 HdT uptake. The methods to identify the cycle phase were: determination of DNA strain content by Feulgen scanning cytophotometry, and determination of chromosome constitution by the technique of premature chromosome condensation (PCC). Preliminary experiments showed the effect of continuous exposure to various levels of 3 HdT on cell growth. High levels of 3 HdT inhibited cell cycle traverse: the cell number and labeling index curves reached a plateau; the cell volume increased; the cells accumulated with 4C DNA contents and it appeared that they blocked in G 2 phase. This pattern is consistent with a radiation effect. (U.S.)

  3. Default cycle phases determined after modifying discrete DNA sequences in plant cells

    International Nuclear Information System (INIS)

    Sans, J.; Leyton, C.

    1997-01-01

    After bromosubstituting DNA sequences replicated in the first, second, or third part of the S phase, in Allium cepa L. meristematic cells, radiation at 313 nm wavelength under anoxia allowed ascription of different sequences to both the positive and negative regulation of some cycle phase transitions. The present report shows that the radiation forced cells in late G 1 phase to advance into S, while those in G 2 remained in G 2 and cells in prophase returned to G 2 when both sets of sequences involved in the positive and negative controls were bromosubstituted and later irradiated. In this way, not only G 2 but also the S phase behaved as cycle phases where cells accumulated by default when signals of different sign functionally cancelled out. The treatment did not halt the rates of replication or transcription of plant bromosubstituted DNA. The irradiation under hypoxia apparently prevents the binding of regulatory proteins to Br-DNA. (author)

  4. G2 phase arrest of cell cycle induced by ionizing radiation

    International Nuclear Information System (INIS)

    Liu Guangwei; Gong Shouliang

    2002-01-01

    The exposure of mammalian cells to X rays results in the prolongation of the cell cycle, including the delay or the arrest in G 1 , S and G 2 phase. The major function of G 1 arrest may be to eliminate the cells containing DNA damage and only occurs in the cells with wild type p53 function whereas G 2 arrest following ionizing radiation has been shown to be important in protecting the cells from death and occurs in all cells regardless of p53 status. So the study on G 2 phase arrest of the cell cycle induced by ionizing radiation has currently become a focus at radiobiological fields

  5. Evolution of cell resistance, threshold voltage and crystallization temperature during cycling of line-cell phase-change random access memory

    NARCIS (Netherlands)

    Oosthoek, J. L. M.; Attenborough, K.; Hurkx, G. A. M.; Jedema, F. J.; Gravesteijn, D. J.; Kooi, B. J.

    2011-01-01

    Doped SbTe phase change (PRAM) line cells produced by e-beam lithography were cycled 100 million times. During cell cycling the evolution of many cell properties were monitored, in particular the crystalline and amorphous resistance, amorphous resistance drift exponent, time-dependent threshold

  6. Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells.

    Science.gov (United States)

    Onumah, Ogbeyalu E; Jules, George E; Zhao, Yanfeng; Zhou, LiChun; Yang, Hong; Guo, ZhongMao

    2009-06-15

    Although it is understood that hydrogen peroxide (H(2)O(2)) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H(2)O(2) in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H(2)O(2). The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0 +/- 4.7 h versus 28.6 +/- 0.8 h, pcatalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G(0)/G(1) phase in hCatTg MAECs compared to the wild-type cells (25.0 +/- 0.9 h versus 15.9 +/- 1.4 h, pinhibitors, p21 and p27, which inhibit the Cdk activity required for the G(0)/G(1)- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H(2)O(2) scavenger, suggest that endogenously produced H(2)O(2) mediates MAEC proliferation by fostering the transition from G(0)/G(1) to S phase.

  7. Cell Cycle Phase Abnormalities Do Not Account for Disordered Proliferation in Barrett's Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Pierre Lao-Sirieix

    2004-11-01

    Full Text Available Barrett's esophagus (BE epithelium is the precursor lesion for esophageal adenocarcinoma. Cell cycle proteins have been advocated as biomarkers to predict the malignant potential in BE. However, whether disruption of the cell cycle plays a causal role in Barrett's carcinogenesis is not clear. Specimens from the Barrett's dysplasia—carcinoma sequence were immunostained for cell cycle phase markers (cyclin D1 for G1; cyclin A for S, G2, and M; cytoplasmic cyclin B1 for G2; and phosphorylated histone 3 for M phase and expressed as a proportion of proliferating cells. Flow cytometric analysis of the cell cycle phase of prospective biopsies was also performed. The proliferation status of nondysplastic BE was similar to gastric antrum and D2, but the proliferative compartment extended to the luminal surface. In dysplastic samples, the number of proliferating cells correlated with the degree of dysplasia (P < .001. The overall levels of cyclins A and B1 correlated with the degree of dysplasia (P < .001. However, the cell cycle phase distribution measured with both immunostaining and flow cytometry was conserved during all stages of BE, dysplasia, and cancer. Hence, the increased proliferation seen in Barrett's carcinogenesis is due to abnormal cell cycle entry or exit, rather than a primary abnormality within the cell cycle.

  8. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

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

  9. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

    International Nuclear Information System (INIS)

    Stamato, T.D.; Weinstein, R.; Giaccia, A.; Mackenzie, L.

    1983-01-01

    A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage

  10. Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

    Science.gov (United States)

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

    2013-07-01

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

  11. Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Daigaku, Yasukazu [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan); Mashiko, Satsuki [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan); Mishiba, Keiichiro [Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003 (Japan); Yamamura, Saburo [Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003 (Japan); Ui, Ayako [Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578 (Japan); Enomoto, Takemi [Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578 (Japan); Yamamoto, Kazuo [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan)]. E-mail: yamamot@mail.tains.tohoku.ac.jp

    2006-08-30

    A CAN1/can1{delta} heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

  12. Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

    Science.gov (United States)

    Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

    2006-08-30

    A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

  13. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Siem van der Laan

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

  14. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

    Science.gov (United States)

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

    2014-01-01

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

  15. Solar Cycle Phase Dependence of Supergranular Fractal Dimension

    Indian Academy of Sciences (India)

    Solar Cycle Phase Dependence of Supergranular Fractal Dimension ... NIE Institute of Technology, Mysore, India. ... This means that each accepted article is being published immediately online with DOI and article citation ID with starting page ...

  16. Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

    Science.gov (United States)

    Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2018-02-01

    Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

  17. Inhibition of G1-phase arrest induced by ionizing radiation in hematopoietic cells by overexpression of genes involved in the G1/S-phase transition

    International Nuclear Information System (INIS)

    Epperly, M.; Berry, L.; Halloran, A.; Greenberger, J.S.

    1995-01-01

    D-type cyclins and cyclin-dependent kinase (cdk-4) are likely involved in regulating passage of cells through the G 1 phase of the cell cycle. A decrease in the proportion of cells in G 1 , a relatively radiation-sensitive phase of the cell cycle, should result in increased resistance to ionizing radiation; however, the effect of such overexpression on X-ray-induced G 1 -phase arrest is not known. Radiation survival curves were obtained at a dose rate of either 8 cGy/min or 1 Gy/min for subclones of the IL-3-dependent hematopoietic progenitor cell line 32D cl 3 expressing transgenes for either cyclin-D1, D2 or D3 or cdk-4. We compared the results to those with overexpression of the transgene for Bcl-2, whose expression enhances radiation survival and delays apoptosis. Cells overexpressing transgenes for each D-type cyclin or Bcl-2 had an increased number of cells in S phase compared to parent line 32D cl 3; however, overexpression of cdk-4 had no effect on cell cycle distribution. Cell death resulting from withdrawal of IL-3 was not affected by overexpression of D2, cdk-4 or Bcl-2. Flow cytometry 24 h after 5 Gy irradiation demonstrated that overexpression of each G 1 -phase regulatory transgene decreased the proportion of cells at the G 1 /S-phase border. Western analysis revealed induction of cyclin-D protein levels by irradiation, but no change in the D O , but a significant increase in the rvec n for cyclin-D or cdk-4 transgene-overexpressing clones at 1 Gy/min (P 1 /S-phase arrest. 31 refs., 4 figs., 4 tabs

  18. Phase-changes in cell cycle of wound tissue irradiated with 5.21 Gy soft X-rays

    International Nuclear Information System (INIS)

    Liu Jianzhong; Zhou Yuanguo; Cheng Tianmin; Zhou Ping; Liu Xia; Li Ping

    2002-01-01

    Objective: To study the phase-changes in cell cycle of wound tissue which was locally irradiated with 5.21 Gy soft X-rays. Methods: Flow cytometry and PI staining were used to analyze cell cycle. Cell proliferation was determined with BrdU labeling. Results: During 3-9 days after irradiation, the percentage of the G 0 /G 1 phase cells in wound of the control side decreased while the percentage of S phase cells increased and reached the highest value on day 9. The percentage of G 2 /M phase cells also increased, and reached its peak on day 15. The percentage of G 0 /G 1 phase cell increased in wound of the irradiation side and was higher than that of the control wound, meanwhile the percentages of S and G 2 /M cells were significantly lower than those of the control wound. In the period of 12-22 days after wounding, the percentage of S phase cells increased and reached its peak value on the 22 th day. When most of cells were in S phase and arrested dramatically. Through the whole healing process, the percentage of G 2 /M in wound of the irradiation side was lower than that of the non-irradiated wound. The BrdU-positive cells were fibroblasts, endothelial cells and smooth muscle cells. Conclusion: These results suggest that G 1 block, S phase arrest, and switch of G 2 /M with suppression of mitotic activity of these cells are induced by local 5.21 Gy soft X-ray irradiation. Therefore, wound healing delay is induced partly by cell cycle arrest

  19. Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

    Science.gov (United States)

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

    2013-01-01

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

  20. Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

    Directory of Open Access Journals (Sweden)

    Yoshinori Kagawa

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

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

    Science.gov (United States)

    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.

  2. Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

    Science.gov (United States)

    Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

    2017-11-22

    Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Cell cycle delays induced by heavy ion irradiation of synchronous mammalian cells

    International Nuclear Information System (INIS)

    Scholz, M.; Kraft-Weyrather, W.; Ritter, S.; Kraft, G.

    1994-01-01

    Cell cycle delays in V79 Chinese hamster cells induced by heavy ion exposure have been investigated using flow cytometry. Synchronous cell populations in G 1 -, S- and late-S/G 2 M-phase were used. Cells were irradiated with particles from Z = 10 (neon) up to Z = 96 (uranium) in the energy range from 2.4 to 17.4 MeV/u and the LET range from 415 to 16225 keV/μm at the UNILAC at GSI, Darmstadt. For comparison, experiments with 250 kV X-rays were performed. For light particles like neon, cell cycle perturbations comparable to those after X-ray irradiation were found, and with increasing LET an increasing delay per particle traversal was observed. For the highest LET-values, extended delays in G 1 -, S- and G 2 M-phase were detected immediately after irradiation. A large fraction of the cells remained in S-phase or G 2 M-phase up to 48 h or longer after irradiation. No significant cell age dependence of cycle delays was detected for the very high LET values. In addition to cell cycle delays, two effects related to the DNA-content as determined by flow cytometry were found after irradiation with very high LET particles, which were attributed to cell fusion and to drastic morphological changes of the cells. Estimations based on the dose deposited by a single particle hit in the cell nucleus and the actual number of hits show, that the basic trend of the experimental results can be explained by the stochastic properties of particle radiation. (orig.)

  4. Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle.

    Science.gov (United States)

    Yamashita, Tomoko; Nishimura, Kazuhiro; Saiki, Ryotaro; Okudaira, Hiroyuki; Tome, Mayuko; Higashi, Kyohei; Nakamura, Mizuho; Terui, Yusuke; Fujiwara, Kunio; Kashiwagi, Keiko; Igarashi, Kazuei

    2013-06-01

    The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  6. Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells.

    Science.gov (United States)

    Takemura, Yukitoshi; Satoh, Motohiko; Hatanaka, Kenichi; Kubota, Shunichiro

    2018-04-24

    Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.

  7. Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

    Science.gov (United States)

    Kaldis, Philipp; Aleem, Eiman

    2005-11-01

    It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings.

  8. Identification of a novel EGF-sensitive cell cycle checkpoint

    International Nuclear Information System (INIS)

    Walker, Francesca; Zhang Huihua; Burgess, Antony W.

    2007-01-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells

  9. Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

    Science.gov (United States)

    Grolmusz, Vince Kornél; Karászi, Katalin; Micsik, Tamás; Tóth, Eszter Angéla; Mészáros, Katalin; Karvaly, Gellért; Barna, Gábor; Szabó, Péter Márton; Baghy, Kornélia; Matkó, János; Kovalszky, Ilona; Tóth, Miklós; Rácz, Károly; Igaz, Péter; Patócs, Attila

    2016-01-01

    Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

  10. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different

  11. Effects of nicotine on cellular proliferation, cell cycle phase distribution, and macromolecular synthesis in human promyelocytic HL-60 leukaemia cells

    International Nuclear Information System (INIS)

    Konno, S.; Wu, J.M.; Chiao, J.W.

    1986-01-01

    Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in the human promyelocytic HL-60 leukemia cells, with 4 mM nicotine resulting in a 50% inhibition of cellular proliferation after 48-50h. Accompanying the anticellular effect of nicotine is a significant change in the cell cycle distribution of HL-60 cells. For example, treatment with 4 mM nicotine for 20h causes an increase in the proportion of G1-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes partial cell arrest in the G-1 phase which may in part account for its effects on cell growth. To determine whether nicotine changes the cellular uptake/transport to macromolecular precursors, HL-60 cells were treated with 216 mM nicotine for 30h, at the end of which time cells were labelled with ( 3 H)thymidine, ( 3 H)uridine, ( 14 C)lysine and( 35 S)methionine, the trichloroacetic acid soluble and insoluble radioactivities from each of the labelling conditions were determined. These studies show that nicotine mainly affects the ''de novo synthesis'' of proteins. (author)

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

    Science.gov (United States)

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

    2016-04-01

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

  13. Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lin [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Yue, Grace G.L. [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Lau, Clara B.S. [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); Sun, Handong [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, CAS, Yunnan (China); Fung, Kwok Pui [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China); Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Leung, Ping Chung [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); Han, Quanbin, E-mail: simonhan@hkbu.edu.hk [Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong (China); State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong (China); School of Chinese Medicine, The Hong Kong Baptist University, Hong Kong (China); Leung, Po Sing, E-mail: psleung@cuhk.edu.hk [School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (China)

    2012-07-01

    Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment. -- Highlights: ► We study Eriocalyxin B (EriB)'s cytotoxic effects on pancreatic cancer cell lines. ► EriB inhibits cell proliferation via mediation of apoptosis and cell cycle arrest. ► The effects are involved in caspase-dependent apoptosis and p53 pathway. ► In vivo study also shows EriB inhibits the growth of human pancreatic tumor. ► EriB can be a good candidate for chemotherapy in pancreatic cancer.

  14. Huaier Aqueous Extract Induces Hepatocellular Carcinoma Cells Arrest in S Phase via JNK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Chengshuo Zhang

    2015-01-01

    Full Text Available Huaier aqueous extract, the main active constituent of Huaier proteoglycan, has antihepatocarcinoma activity in experimental and clinical settings. However, the potential and associated antihepatoma mechanisms of Huaier extract are not yet fully understood. Therefore, in this study, we aimed to elucidate the inhibitory proliferation effect of Huaier extract on apoptosis and cycle of HepG2 and Bel-7402 cells. Our data demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability dose-dependently. Flow cytometric analysis showed that a 48 h treatment of Huaier extract caused cell apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after Hoechst staining. Immunoblot analysis further demonstrated that Huaier extract activated caspase 3 and PARP. Additionally, Huaier extract inhibited the activity of p-ERK, p-p38, and p-JNK in terms of MAPK. Furthermore, Huaier extract induced HCC cells arrest in S phase and decreased the cycle related protein expression of β-catenin and cyclin D1. Studies with JNK specific inhibitor, SP600125, showed that Huaier extract induced S phase arrest and decreased β-catenin and cyclin D1 expression via JNK signaling pathway. In conclusion, we verify that Huaier extract causes cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management.

  15. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  16. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

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

  17. Alteration of cell cycle progression by Sindbis virus infection

    International Nuclear Information System (INIS)

    Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

    2015-01-01

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

  18. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    Science.gov (United States)

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  19. Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

    Directory of Open Access Journals (Sweden)

    Selene Glück

    Full Text Available In infected cells rotavirus (RV replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2 involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.

  20. RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle.

    Science.gov (United States)

    Yamane, Arito; Robbiani, Davide F; Resch, Wolfgang; Bothmer, Anne; Nakahashi, Hirotaka; Oliveira, Thiago; Rommel, Philipp C; Brown, Eric J; Nussenzweig, Andre; Nussenzweig, Michel C; Casellas, Rafael

    2013-01-31

    Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  1. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-01-07

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

  3. The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

    Science.gov (United States)

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

    2017-08-01

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

  4. High content image based analysis identifies cell cycle inhibitors as regulators of Ebola virus infection.

    Science.gov (United States)

    Kota, Krishna P; Benko, Jacqueline G; Mudhasani, Rajini; Retterer, Cary; Tran, Julie P; Bavari, Sina; Panchal, Rekha G

    2012-09-25

    Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV) infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics.

  5. High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

    Directory of Open Access Journals (Sweden)

    Sina Bavari

    2012-09-01

    Full Text Available Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics.

  6. Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

    Science.gov (United States)

    Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

    2017-11-06

    Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  7. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Yamamoto, Ryohei; Ohshiro, Yukari; Shimotani, Tatsuhiko; Yamamoto, Mizuki; Matsuyama, Satoshi; Ide, Hiroshi; Kubo, Kihei

    2014-01-01

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H 2 O 2 ) sensitivity. Hypersensitivity to H 2 O 2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

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

    Science.gov (United States)

    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.

  9. Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F

    DEFF Research Database (Denmark)

    Hateboer, G; Wobst, A; Petersen, B O

    1998-01-01

    The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have...... of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells...

  10. Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Bergdahl, Andreas; Christophersen, Palle

    2007-01-01

    Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored...... during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current......+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle...

  11. Cell growth and division cycle

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.

    1986-01-01

    The concept of the cell cycle in its present form was introduced more than three decades ago. Studying incorporation of DNA precursors by autoradiography, these authors observed that DNA synthesis in individual cells was discontinuous and occupied a discrete portion of the cell life (S phase). Mitotic division was seen to occur after a certain period of time following DNA replication. A distinct time interval between mitosis and DNA replication was also apparent. Thus, the cell cycle was subdivided into four consecutive phases, G/sub 1/, S, G/sub 2/, and M. The G/sub 1/ and G/sub 2/ phases represented the ''gaps'' between mitosis and the start of DNA replication, and between the end of DNA replication and the onset of mitosis, respectively. The cell cycle was defined as the interval between the midpoint of mitosis and the midpoint of the subsequent mitosis of the daughter cell(s). The authors' present knowledge on the cell cycle benefited mostly from the development of four different techniques: autoradiography, time-lapse cinematography, cell synchronization and flow cytometry. Of these, autoradiography has been the most extensively used, especially during the past two decades. By providing a means to analyse incorporation of precursors of DNA, RNA or proteins by individual cells and, in combination with various techniques of cell synchronization, autoradiography yielded most of the data fundamental to the current understanding of the cell cycle-related phenomena. Kinetics of cell progression through the cell cycle could be analysed in great detail after development of such sophisticated autoradiographic approaches as measurements of the fraction of labeled mitoses (''FLM curves'') or multiple sequential cell labelling with /sup 3/H- and /sup 14/C-TdR

  12. Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

    Science.gov (United States)

    Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

    2008-01-01

    The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

  13. Cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bootsma, A.L. (Rijksuniversiteit Utrecht (Netherlands). Academisch Ziekenhuis); Davids, J.A.G. (Netherlands Energy Research Foundation, Petten (Netherlands))

    1988-03-01

    In the CBA mouse testis, about 10% of the stem cell population is highly resistant to neutron irradiation (D/sub 0/, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea, it appeared that in this cycle the S-phase is less radiosensitive (D/sub 0/, 0.43 Gy) than the other phases of the cell cycle (D/sub 0/, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation, the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most. (author).

  14. Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1

    International Nuclear Information System (INIS)

    Kang, Du-Seock; Hong, Kyeong-Man; Park, Joobae; Bae, Chang-Dae

    2012-01-01

    Highlights: ► We identified a GC box and a CHR element in human CKAP2 minimal promoter. ► The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. ► The GC box was essential for the basic promoter activity of human CKAP2. ► The GC box was also essential for the cyclic expression of human CKAP2. ► The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (−110 to −104 bp) and a GC box (−41 to −32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

  15. The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle

    Science.gov (United States)

    Scherer, Yvette D.

    2014-01-01

    In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

  16. Changes in distribution of cell cycle phases and DNA content in HeLa S3 cell after irradiation

    International Nuclear Information System (INIS)

    Wang Shunbao

    1992-01-01

    The effects of irradiation and hyperthermia on the distribution in various phases and DNA content of HeLa S 3 cells were analyzed by flow cytometry and an image analysis instrument. A marked increase in DNA content from 6.718 to 9.614(AU) in HeLa S 3 cells after 6 Gy irradiation was seen to correspond with the changes in the distribution of various phases in G 2 + M, from 22% to 52%. Meanwhile, the surviving fraction of HeLa S 3 cells after 6 Gy irradiation was less than 1%. However, after heating at 44 deg C for 10 min, the amount of cells in G 2 + M increased from 22.5% to 52.5% and the surviving fraction after hyperthermia was less than 2.65%. The changes in distribution of various phases after Ir-192 irradiation were similar to those seen after X-ray irradiation. The delay of G 2 + M phase after treatment with 8 Gy plus heating at 44 deg C for 7 min in HeLa S 3 cells was similar to that seen in the case of treatment with 8 Gy alone. As the surviving fraction accompanying the G 2 + M delay after irradiation plus heat treatment was very low, we suggest that the changes of distribution in various phases of HeLa S 3 cells after treatment might be used as a rapid indicator of serious injury

  17. Curcumin analog WZ35 induced cell death via ROS-dependent ER stress and G2/M cell cycle arrest in human prostate cancer cells

    International Nuclear Information System (INIS)

    Zhang, Xiuhua; Chen, Minxiao; Zou, Peng; Kanchana, Karvannan; Weng, Qiaoyou; Chen, Wenbo; Zhong, Peng; Ji, Jiansong; Zhou, Huiping; He, Langchong; Liang, Guang

    2015-01-01

    Prostate cancer is the most commonly diagnosed malignancy among men. The Discovery of new agents for the treatment of prostate cancer is urgently needed. Compound WZ35, a novel analog of the natural product curcumin, exhibited good anti-prostate cancer activity, with an IC 50 of 2.2 μM in PC-3 cells. However, the underlying mechanism of WZ35 against prostate cancer cells is still unclear. Human prostate cancer PC-3 cells and DU145 cells were treated with WZ35 for further proliferation, apoptosis, cell cycle, and mechanism analyses. NAC and CHOP siRNA were used to validate the role of ROS and ER stress, respectively, in the anti-cancer actions of WZ35. Our results show that WZ35 exhibited much higher cell growth inhibition than curcumin by inducing ER stress-dependent cell apoptosis in human prostate cells. The reduction of CHOP expression by siRNA partially abrogated WZ35-induced cell apoptosis. WZ35 also dose-dependently induced cell cycle arrest in the G2/M phase. Furthermore, we found that WZ35 treatment for 30 min significantly induced reactive oxygen species (ROS) production in PC-3 cells. Co-treatment with the ROS scavenger NAC completely abrogated the induction of WZ35 on cell apoptosis, ER stress activation, and cell cycle arrest, indicating an upstream role of ROS generation in mediating the anti-cancer effect of WZ35. Taken together, this work presents the novel anticancer candidate WZ35 for the treatment of prostate cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human prostate cancer treatment. The online version of this article (doi:10.1186/s12885-015-1851-3) contains supplementary material, which is available to authorized users

  18. Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

    Science.gov (United States)

    Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

    2013-01-01

    The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

  19. The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

    Science.gov (United States)

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

    2008-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

    2014-08-01

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

  1. Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways

    International Nuclear Information System (INIS)

    Li, Lin; Yue, Grace G.L.; Lau, Clara B.S.; Sun, Handong; Fung, Kwok Pui; Leung, Ping Chung; Han, Quanbin; Leung, Po Sing

    2012-01-01

    Pancreatic cancer is difficult to detect early and responds poorly to chemotherapy. A breakthrough in the development of new therapeutic agents is urgently needed. Eriocalyxin B (EriB), isolated from the Isodon eriocalyx plant, is an ent-kaurane diterpenoid with promise as a broad-spectrum anti-cancer agent. The anti-leukemic activity of EriB, including the underlying mechanisms involved, has been particularly well documented. In this study, we demonstrated for the first time EriB's potent cytotoxicity against four pancreatic adenocarcinoma cell lines, namely PANC-1, SW1990, CAPAN-1, and CAPAN-2. The effects were comparable to that of the chemotherapeutic camptothecin (CAM), but with much lower toxicity against normal human liver WRL68 cells. EriB's cytoxicity against CAPAN-2 cells was found to involve caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Moreover, the p53 pathway was found to be activated by EriB in these cells. Furthermore, in vivo studies showed that EriB inhibited the growth of human pancreatic tumor xenografts in BALB/c nude mice without significant secondary adverse effects. These results suggest that EriB should be considered a candidate for pancreatic cancer treatment. -- Highlights: ► We study Eriocalyxin B (EriB)'s cytotoxic effects on pancreatic cancer cell lines. ► EriB inhibits cell proliferation via mediation of apoptosis and cell cycle arrest. ► The effects are involved in caspase-dependent apoptosis and p53 pathway. ► In vivo study also shows EriB inhibits the growth of human pancreatic tumor. ► EriB can be a good candidate for chemotherapy in pancreatic cancer.

  2. Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-dependent pathway in human colon cancer cells.

    Science.gov (United States)

    Zhang, Zhiyu; Wang, Chong-Zhi; Du, Guang-Jian; Qi, Lian-Wen; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

    2013-07-01

    Soybean isoflavones have been used as a potential preventive agent in anticancer research for many years. Genistein is one of the most active flavonoids in soybeans. Accumulating evidence suggests that genistein alters a variety of biological processes in estrogen-related malignancies, such as breast and prostate cancers. However, the molecular mechanism of genistein in the prevention of human colon cancer remains unclear. Here we attempted to elucidate the anticarcinogenic mechanism of genistein in human colon cancer cells. First we evaluated the growth inhibitory effect of genistein and two other isoflavones, daidzein and biochanin A, on HCT-116 and SW-480 human colon cancer cells. In addition, flow cyto-metry was performed to observe the morphological changes in HCT-116/SW-480 cells undergoing apoptosis or cell cycle arrest, which had been visualized using Annexin V-FITC and/or propidium iodide staining. Real-time PCR and western blot analyses were also employed to study the changes in expression of several important genes associated with cell cycle regulation. Our data showed that genistein, daidzein and biochanin A exhibited growth inhibitory effects on HCT-116/SW-480 colon cancer cells and promoted apoptosis. Genistein showed a significantly greater effect than the other two compounds, in a time- and dose-dependent manner. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of ATM/p53, p21waf1/cip1 and GADD45α as well as downregulation of cdc2 and cdc25A demonstrated by q-PCR and immunoblotting assay. Interestingly, genistein induced G2/M cell cycle arrest in a p53-dependent manner. These findings exemplify that isoflavones, especially genistein, could promote colon cancer cell growth inhibition and facilitate apoptosis and cell cycle arrest in the G2/M phase. The ATM/p53-p21 cross-regulatory network may play a crucial role in mediating the anticarcinogenic activities of genistein in colon cancer.

  3. SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Cao, Rubo; Ding, Qian; Li, Pindong; Xue, Jun; Zou, Zhenwei; Huang, Jing; Peng, Gang

    2013-01-01

    Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

  4. Late phase cell cycle proteins in Alzheimer’s disease: a possible target for therapy?

    KAUST Repository

    Bajic, Vladan

    2017-02-22

    Alzheimer’s disease (AD) is represented by neuronal loss and this loss is correlated to a constant state of neuronal instability induced by intrinsic and extrinsic factors. In this paper data is presented regarding the possible roles of late phase cell cycle proteins in normal and affected neurons with the goal that understanding the mechanisms involved in the regulation of these proteins may represent a novel strategy for AD treatment. The results demonstrate a relative differential pattern of expression of certain proteins (APC/C, Mad1 and Mad2, Bub R1, Bub1, CDK 11, cohesin subunit Rad 21 and astrin) in the AD brain versus age matched controls, and it is suggested that targeting these proteins might translate into potential treatments for AD. Although the data presented here is of some interest, the ability to translate such information into clinical applications is often a challenge.

  5. Late phase cell cycle proteins in Alzheimer’s disease: a possible target for therapy?

    KAUST Repository

    Bajic, Vladan; B. Bajic, Vladimir; Zivkovic, Lada; Arendt, Thomas; Perry, George; Spremo-Potparevic, Biljana

    2017-01-01

    Alzheimer’s disease (AD) is represented by neuronal loss and this loss is correlated to a constant state of neuronal instability induced by intrinsic and extrinsic factors. In this paper data is presented regarding the possible roles of late phase cell cycle proteins in normal and affected neurons with the goal that understanding the mechanisms involved in the regulation of these proteins may represent a novel strategy for AD treatment. The results demonstrate a relative differential pattern of expression of certain proteins (APC/C, Mad1 and Mad2, Bub R1, Bub1, CDK 11, cohesin subunit Rad 21 and astrin) in the AD brain versus age matched controls, and it is suggested that targeting these proteins might translate into potential treatments for AD. Although the data presented here is of some interest, the ability to translate such information into clinical applications is often a challenge.

  6. Multiparameter Cell Cycle Analysis.

    Science.gov (United States)

    Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

    2018-01-01

    Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

  7. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pearlman, Andrew Leonard [Univ. of California, Berkeley, CA (United States)

    1978-08-01

    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G2M by about 50%. When added to G1 cells, DE delayed recruitment of apparently quiescent (G0) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  8. Apoptosis Induction by Polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and S-phase cell cycle arrest in HepG2 cell line.

    Science.gov (United States)

    Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

    2014-01-01

    Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

  9. Chalepin: A Compound from Ruta angustifolia L. Pers Exhibits Cell Cycle Arrest at S phase, Suppresses Nuclear Factor-Kappa B (NF-κB) Pathway, Signal Transducer and Activation of Transcription 3 (STAT3) Phosphorylation and Extrinsic Apoptotic Pathway in Non-small Cell Lung Cancer Carcinoma (A549).

    Science.gov (United States)

    Richardson, Jaime Stella Moses; Aminudin, Norhaniza; Abd Malek, Sri Nurestri

    2017-10-01

    Plants have been a major source of inspiration in developing novel drug compounds in the treatment of various diseases that afflict human beings worldwide. Ruta angustifolia L. Pers known locally as Garuda has been conventionally used for various medicinal purposes such as in the treatment of cancer. A dihydrofuranocoumarin named chalepin, which was isolated from the chloroform extract of the plant, was tested on its ability to inhibit molecular pathways of human lung carcinoma (A549) cells. Cell cycle analysis and caspase 8 activation were conducted using a flow cytometer, and protein expressions in molecular pathways were determined using Western blot technique. Cell cycle analysis showed that cell cycle was arrested at the S phase. Further studies using Western blotting technique showed that cell cycle-related proteins such as cyclins, cyclin-dependent kinases (CDKs), and inhibitors of CDKs correspond to a cell cycle arrest at the S phase. Chalepin also showed inhibition in the expression of inhibitors of apoptosis proteins. Nuclear factor-kappa B (NF-κB) pathway, signal transducer and activation of transcription 3 (STAT-3), cyclooxygenase-2, and c-myc were also downregulated upon treatment with chalepin. Chalepin was found to induce extrinsic apoptotic pathway. Death receptors 4 and 5 showed a dramatic upregulation at 24 h. Analysis of activation of caspase 8 with the flow cytometer showed an increase in activity in a dose- and time-dependent manner. Activation of caspase 8 induced cleavage of BH3-interacting domain death agonist, which initiated a mitochondrial-dependent or -independent apoptosis. Chalepin causes S phase cell cycle arrest, NF-κB pathway inhibition, and STAT-3 inhibition, induces extrinsic apoptotic pathway, and could be an excellent chemotherapeutic agent. This study reports the capacity of an isolated bioactive compound known as chalepin to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, signal

  10. Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

    Science.gov (United States)

    Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

    2018-04-01

    Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

  11. Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation

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    Lin Chih-Li

    2010-02-01

    Full Text Available Abstract Background Functional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood. Methods & Results Here we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities. Conclusions Together, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.

  12. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

    Science.gov (United States)

    Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

    2011-06-14

    The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

  13. Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle

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    Gabriele Büchel

    2017-12-01

    Full Text Available MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II. To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

  14. Effects of arsenite on cell cycle progression in a human bladder cancer cell line

    International Nuclear Information System (INIS)

    Hernandez-Zavala, A.; Cordova, E.; Razo, L.M. del; Cebrian, M.E.; Garrido, E.

    2005-01-01

    Bladder cancer is one of the most important diseases associated with arsenic (As) exposure in view of its high prevalence and mortality rate. Experimental studies have shown that As exposure induces cell proliferation in the bladder of sodium arsenite (iAsIII) subchronically treated mice. However, there is little available information on its effects on the cell cycle of bladder cells. Thus, our purpose was to evaluate the effects of iAsIII on cell cycle progression and the response of p53 and p21 on the human-derived epithelial bladder cell line HT1197. iAsIII treatment (1-10 μM) for 24 h induced a dose-dependent increase in the proportion of cells in S-phase, which reached 65% at the highest dose. A progressive reduction in cell proliferation was also observed. BrdU was incorporated to cellular DNA in an interrupted form, suggesting an incomplete DNA synthesis. The time-course of iAsIII effects (10 μM) showed an increase in p53 protein content and a transient increase in p21 protein levels accompanying the changes in S-phase. These effects were correlated with iAs concentrations inside the cells, which were not able to metabolize inorganic arsenic. Our findings suggest that p21 was not able to block CDK2-cyclin E complex activity and was therefore unable to arrest cells in G1 allowing their progression into the S-phase. Further studies are needed to ascertain the mechanisms underlying the effects of iAsIII on the G1 to S phase transition in bladder cells

  15. Allyl isothiocyanate affects the cell cycle of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Signe Elisabeth Åsberg

    2015-05-01

    Full Text Available Isothiocyanates (ITCs are degradation products of glucosinolates present in members of the Brassicaceae family acting as herbivore repellents and antimicrobial compounds. Recent results indicate that allyl ITC (AITC has a role in defense responses such as glutathione depletion, ROS generation and stomatal closure. In this study we show that exposure to non-lethal concentrations of AITC causes a shift in the cell cycle distribution of Arabidopsis thaliana leading to accumulation of cells in S-phases and a reduced number of cells in non-replicating phases. Furthermore, transcriptional analysis revealed an AITC-induced up-regulation of the gene encoding cyclin-dependent kinase A while several genes encoding mitotic proteins were down-regulated, suggesting an inhibition of mitotic processes. Interestingly, visualization of DNA synthesis indicated that exposure to AITC reduced the rate of DNA replication. Taken together, these results indicate that non-lethal concentrations of AITC induce cells of A. thaliana to enter the cell cycle and accumulate in S-phases, presumably as a part of a defensive response. Thus, this study suggests that AITC has several roles in plant defense and add evidence to the growing data supporting a multifunctional role of glucosinolates and their degradation products in plants.

  16. Cell cycle analysis of brain cells as a growth index in larval cod at different feeding conditions and temperatures

    Directory of Open Access Journals (Sweden)

    Rafael González-Quirós

    2007-09-01

    Full Text Available The percentage of cells dividing in a specific tissue of individual larvae can be estimated by analyzing DNA per cell by flow cytometry. An experimental test was carried out with cod (Gadus morhua larvae, with brain as the target tissue, to validate this technique as an appropriate growth index for larval fish. Standard length (SL, myotome height, and %S-phase (% of cells in the S-phase of the cell-division cycle variability were analyzed, with temperature (6 and 10°C, food level (high- and no-food and larval developmental stage (first feeding, pre-metamorphosis and post-metamorphosis as independent factors. Cod larvae grew faster (in SL and presented a higher %S-phase under high-food conditions. Larval SL increased with temperature in rearing and experimental tanks. However, there was a significant interaction between temperature and food in the %S-phase. There were no significant differences in the %S-phase between 6 and 10°C at high-food levels. We suggest that this result is a consequence of temperature-dependency of the duration of the cell cycle. In the absence of food, larvae at 10ºC had a lower %S-phase than larvae at 6°C, which may be related to increased metabolic costs with increasing temperature. Considering the effect of temperature, the mean % S-phase explained 74% of the variability in the estimated standard growth rate.

  17. p53 represses autophagy in a cell cycle-dependent fashion.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, Maria Chiara; Orhon, Idil; Kepp, Oliver; Morselli, Eugenia; Criollo, Alfredo; Kroemer, Guido

    2008-10-01

    Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-alpha), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-X(L) with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.

  18. Cell cycle deregulation by the HBx protein of hepatitis B virus

    Directory of Open Access Journals (Sweden)

    Vijay Kumar

    2007-02-01

    Full Text Available

    Cell cycle control by oncogenic viruses usually involves disruption of the normal restraints on cellular proliferation via abnormal proteolytic degradation and malignant transformation of cells. The cell cycle regulatory molecules viz. cyclins, cyclin-dependent kinases (cdks and inhibitors of cdks as well as the transcriptional targets of signaling pathways induce cells to move through the cell cycle checkpoints. These check points are often found deregulated in tumor cells and in the cells afflicted with DNA tumor viruses predisposing them towards transformation. The X protein or HBx of hepatitis B virus is a promiscuous transactivator that has been implicated in the development of hepatocellular carcinoma in humans. However, the exact role of HBx in establishing a permissive environment for hepatocarcinogenesis is not fully understood. HBx activates the Ras-Raf-MAP kinase signaling cascade, through which it activates transcription factors AP-1 and NFkappa B, and stimulates cell DNA synthesis. HBx shows a profound effect on cell cycle progression even in the absence of serum. It can override the replicative senescence of cells in G0 phase by binding to p55sen. It stimulates the G0 cells to transit through G1 phase by activating Src kinases and the cyclin A-cyclin-dependent kinase 2 complexes, that in turn induces the cyclin A promoter. There is an early and sustained level of cyclin-cdk2 complex in the presence of HBx during the cell cycle which is coupled with an increased protein kinase activity of cdk2 suggesting an early appearance of S phase. The interaction between cyclin-cdk2 complex and HBx occurs through its carboxyterminal region (amino acids 85-119 and requires a constitutive Src kinase activity. The increased cdk2 activity is associated with stabilization of cyclin E as well as proteasomal degradation of cdk inhibitor p27Kip1. Notably, the HBx mutant

  19. Study of the cell cycle control for human malignant mesothelioma lines. Interferon and radiations effect

    International Nuclear Information System (INIS)

    Vivo, C.

    1999-01-01

    In order to better understand the inhibition mechanisms of the IFN-R-HU on tumoral development, the IFN-R-U effect on MM lines has been studied. Three groups of lines has been distinguished: eight sensitive lines, two intermediate and three resistant. The sensitive lines showed a triple locking of the cell cycle: in phases S, G1 and G2. The study of the cell cycle control points function, realized by the MM lines radiation exposure showed the points function on G1/S and-or on G2/M and the dependence or non dependence of the cycle stop of the protein P53 and P21 W at F1/CIP1. (A.L.B.)

  20. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  1. Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

    Science.gov (United States)

    Haass, Nikolas K; Gabrielli, Brian

    2017-07-01

    The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

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    Xi Yu

    2015-01-01

    Full Text Available The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM, respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

  3. Cell cycle checkpoints: reversible when possible, irreversible when needed

    NARCIS (Netherlands)

    Krenning, L.

    2015-01-01

    Cell cycle checkpoints are reversible in nature, and can prevent progression into the next cell cycle phase if needed. In the case of DNA damage, cells can prevent progression from G1 into S phase, and from G2 into mitosis in the presence of DNA double strand breaks. Following DNA repair, these

  4. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  5. Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

    Science.gov (United States)

    Saqcena, M; Mukhopadhyay, S; Hosny, C; Alhamed, A; Chatterjee, A; Foster, D A

    2015-05-14

    Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

  6. Dose-rate effects on the cell cycle and survival of S3 HeLa and V79 cells

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Bedford, J.S.; Bailey, S.M.

    1979-01-01

    The effects of continuous irradiation at different dose rates on the cell cycle and on cell survival were studied using synchronized S3 HeLa and V79 cells. The minimum dose rate necessary to stop cell division was found to be approximately 23 rad/hr for HeLa cells and 270 rad/hr for V79 cells. For dose rates that stop cell division, cells progress through G 1 and S, with a small delay in the S phase, and are blocked in G 2 . Appreciable mitotic accumulation was observed for HeLa cells at dose rates which stopped cell division. By comparison, much less mitotic accumulation was observed for V79 cells over a range of dose rates from 37 to 270 rad/hr. Minimum mitotic delays for a variety of dose rates were determined for both cell lines. S3 HeLa cells are much more sensitive in this respect than V79 cells; however, it appeared that for higher dose rates the minimum mitotic delay in HeLa cells asymptotically approached a value of about 35 hr. In addition to the qualitative differences observed for the two cell lines in regard to mitotic accumulation, HeLa cells accumulated for prolonged periods in the presence of colcemid while V79 cells were blocked for only a few hours, HeLa cells show a dramatic effect of redistribution of cells into sensitive phases of the cell cycle during exposure, which was reflected in the survival curves at low dose rate. More cell killing per unit dose was observed at 37 than at 74 rad/hr

  7. Cell cycle arrest induced by radiation

    International Nuclear Information System (INIS)

    Okaichi, Yasuo; Matsumoto, Hideki; Ohnishi, Takeo

    1994-01-01

    It is known that various chemical reactions, such as cell cycle arrest, DNA repair and cell killing, can occur within the cells when exposed to ionizing radiation and ultraviolet radiation. Thus protein dynamics involved in such chemical reactions has received considerable attention. In this article, cell cycle regulation is first discussed in terms of the G2/M-phase and the G1/S-phase. Then, radiation-induced cell cycle arrest is reviewed. Cell cycle regulation mechanism involved in the G2 arrest, which is well known to occur when exposed to radiation, has recently been investigated using yeasts. In addition, recent study has yielded a noticeable finding that the G1 arrest can occur with intracellular accumulation of p53 product following ionization radiation. p53 is also shown to play an extremely important role in both DNA repair and cell killing due to DNA damage. Studies on the role of genes in protein groups induced by radiation will hold promise for the elucidation of cell cycle mechanism. (N.K.) 57 refs

  8. 2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.

    OpenAIRE

    Andreassen, P R; Margolis, R L

    1992-01-01

    BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subs...

  9. A LuxS-Dependent Cell-to-Cell Language Regulates Social Behavior and Development in Bacillus subtilis

    OpenAIRE

    Lombardía, Esteban; Rovetto, Adrián J.; Arabolaza, Ana L.; Grau, Roberto R.

    2006-01-01

    Cell-to-cell communication in bacteria is mediated by quorum-sensing systems (QSS) that produce chemical signal molecules called autoinducers (AI). In particular, LuxS/AI-2-dependent QSS has been proposed to act as a universal lexicon that mediates intra- and interspecific bacterial behavior. Here we report that the model organism Bacillus subtilis operates a luxS-dependent QSS that regulates its morphogenesis and social behavior. We demonstrated that B. subtilis luxS is a growth-phase-regula...

  10. S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons.

    Science.gov (United States)

    Ye, Weizhen; Blain, Stacy W

    2010-08-01

    A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad

  11. Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chang-Jie

    2010-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. Methods Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR and protein (Western blot levels. The phosphorylation status of cyclin-dependent kinases (CDKs and retinoblastoma (Rb protein was also examined using Western blot analysis. Results Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1, pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. Conclusion Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

  12. The role of cell cycle in retinal development: cyclin-dependent kinase inhibitors co-ordinate cell-cycle inhibition, cell-fate determination and differentiation in the developing retina.

    Science.gov (United States)

    Bilitou, Aikaterini; Ohnuma, Shin-ichi

    2010-03-01

    The mature retina is formed through multi-step developmental processes, including eye field specification, optic vesicle evagination, and cell-fate determination. Co-ordination of these developmental events with cell-proliferative activity is essential to achieve formation of proper retinal structure and function. In particular, the molecular and cellular dynamics of the final cell cycle significantly influence the identity that a cell acquires, since cell fate is largely determined at the final cell cycle for the production of postmitotic cells. This review summarizes our current understanding of the cellular mechanisms that underlie the co-ordination of cell-cycle and cell-fate determination, and also describes a molecular role of cyclin-dependent kinase inhibitors (CDKIs) as co-ordinators of cell-cycle arrest, cell-fate determination and differentiation. Copyright (c) 2010 Wiley-Liss, Inc.

  13. Evidence that progression of cells into S-phase is not a prerequisite for recovery between split doses of U.V.-light in synchronized and plateau phase cultures of Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1982-01-01

    The ability of Ehrlich ascites tumour cells (EAT-cells) to perform split-dose recovery after U.V. exposure was studied with unfed plateau phase as well as with synchronized cells selected from exponentially growing cultures. The cells were kept in balanced salt solution which inhibited the progression of the cells through the cell cycle. The results indicated that split-dose recovery occurred in EAT-cells in all phases of the cell cycle and that progression of the cells into S-phase was not a prerequisite for this type of repair. The second-dose survival curves of G 1 -and S-phase cells showed, 24 hours after the first U.V. exposure, a shoulder width comparable to that of singly irradiated cells. Second-dose survival curves for G 2 -cells showed, after the same time interval, a shoulder width smaller than that for singly exposed cells, presumably due to some cell division. The recovery time constant (t 50 between 4 and 8 hours) increased with increasing U.V. exposure. (author)

  14. A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0/G1 cell-cycle arrest

    International Nuclear Information System (INIS)

    Ujiki, Michael B.; Milam, Ben; Ding Xianzhong; Roginsky, Alexandra B.; Salabat, M. Reza; Talamonti, Mark S.; Bell, Richard H.; Gu Wenxin; Silverman, Richard B.; Adrian, Thomas E.

    2006-01-01

    Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Sansalvamide A is a cyclic depsipeptide produced by a marine fungus. We investigated the effect of a novel sansalvamide A analogue on growth, cell-cycle phases, and induction of apoptosis in human pancreatic cancer cells in vitro. The sansalvamide analogue caused marked time- and concentration-dependent inhibition of DNA synthesis and cell proliferation of two human pancreatic cancer cell lines (AsPC-1 and S2-013). The analogue induced G0/G1 phase cell-cycle arrest and morphological changes suggesting induction of apoptosis. Apoptosis was confirmed by annexin V binding. This novel sansalvamide analogue inhibits growth of pancreatic cancer cells through G0/G1 arrest and induces apoptosis. Sansalvamide analogues may be valuable for the treatment of pancreatic cancer

  15. Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by light intensity

    Czech Academy of Sciences Publication Activity Database

    Vítová, Milada; Bišová, Kateřina; Umysová, Dáša; Hlavová, Monika; Kawano, S.; Zachleder, Vilém; Čížková, Mária

    2011-01-01

    Roč. 233, č. 1 (2011), s. 75-86 ISSN 0032-0935 R&D Projects: GA AV ČR IAA500200614; GA ČR GA525/09/0102; GA ČR GA204/09/0111 Institutional research plan: CEZ:AV0Z50200510 Keywords : Cell division timing * Cell cycle phases * Chlamydomonas Subject RIV: EE - Microbiology, Virology Impact factor: 3.000, year: 2011

  16. Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective or radiosensitizing effects as elucidated by the PCC and G2-assay

    Energy Technology Data Exchange (ETDEWEB)

    Sebastià, N., E-mail: natividad.sebastia@uv.es [Radiation Protection Service, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Montoro, A. [Radiation Protection Service, Universitary and Politechnic Hospital La Fe, Valencia (Spain); Grupo de Investigación Biomédica en Imagen GIBI230, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética Clínica, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Hervás, D. [Biostatistics Unit, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Pantelias, G.; Hatzi, V.I. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, National Centre for Scientific Research “Demokritos”, Aghia Paraskevi, Athens (Greece); Soriano, J.M. [Grupo de Investigación Biomédica en Imagen GIBI230, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética Clínica, IIS La Fe, Health Research Institute La Fe, Valencia (Spain); Department of Preventive Medicine and Public Health, Faculty of Pharmacy, University of Valencia, Burjassot, Valencia (Spain); Villaescusa, J.I. [Radiation Protection Service, Universitary and Politechnic Hospital La Fe, Valencia (Spain); and others

    2014-08-15

    Highlights: • Curcumin and trans-resveratrol can exert radioprotective or radiosensitizing effects. • The mechanisms underlying such dual action were elucidated using the PCC and G2-assay. • Radioprotection occurs in non-cycling cells exposed to curcumin and resveratrol. • Radiosensitization occurs in cycling cells exposed to the chemicals. • G2-checkpoint abrogation by the chemicals underlies the radiosensitizing mechanism. - Abstract: Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140 μM curcumin and 2.2 to 220 μM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity

  17. Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective or radiosensitizing effects as elucidated by the PCC and G2-assay

    International Nuclear Information System (INIS)

    Sebastià, N.; Montoro, A.; Hervás, D.; Pantelias, G.; Hatzi, V.I.; Soriano, J.M.; Villaescusa, J.I.

    2014-01-01

    Highlights: • Curcumin and trans-resveratrol can exert radioprotective or radiosensitizing effects. • The mechanisms underlying such dual action were elucidated using the PCC and G2-assay. • Radioprotection occurs in non-cycling cells exposed to curcumin and resveratrol. • Radiosensitization occurs in cycling cells exposed to the chemicals. • G2-checkpoint abrogation by the chemicals underlies the radiosensitizing mechanism. - Abstract: Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140 μM curcumin and 2.2 to 220 μM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity

  18. STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.

    Directory of Open Access Journals (Sweden)

    Pankaj Goyal

    Full Text Available BACKGROUND: Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs, and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a leading to G(1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays. CONCLUSION/SIGNIFICANCE: The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear

  19. ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

    DEFF Research Database (Denmark)

    Høj, Berit Rahbek; la Cour, Peter Jonas Marstrand; Mollerup, Jens

    2009-01-01

    downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate...... that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.......ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2...

  20. Overexpression of cell cycle regulator CDCA3 promotes oral cancer progression by enhancing cell proliferation with prevention of G1 phase arrest

    International Nuclear Information System (INIS)

    Uchida, Fumihiko; Uzawa, Katsuhiro; Kasamatsu, Atsushi; Takatori, Hiroaki; Sakamoto, Yosuke; Ogawara, Katsunori; Shiiba, Masashi; Tanzawa, Hideki; Bukawa, Hiroki

    2012-01-01

    Cell division cycle associated 3 (CDCA3), part of the Skp1-cullin-F-box (SCF) ubiquitin ligase, refers to a trigger of mitotic entry and mediates destruction of the mitosis inhibitory kinase. Little is known about the relevance of CDCA3 to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of CDCA3 in OSCC. We evaluated CDCA3 mRNA and protein expression in both OSCC-derived cell lines and primary OSCCs and performed functional analyses of CDCA3 in OSCC-derived cells using the shRNA system. The CDCA3 expression at both the mRNA and protein levels was frequently up-regulated in all cell lines examined and primary tumors (mRNA, 51/69, 74 %; protein, 79/95, 83 %) compared to normal controls (p < 0.001). In contrast, no significant level of CDCA3 protein expression was seen in oral premalignant lesions (OPLs) (n = 20) compared with the expression in OSCCs. Among the clinical variables analyzed, the CDCA3 expression status was closely related to tumor size (p < 0.05). In addition, suppression of CDCA3 expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase. Further, there was up-regulation of the cyclin-dependent kinase inhibitors (p21 Cip1 , p27 Kip1 , p15 INK4B , and p16 INK4A ) in the knockdown cells. The current results showed that overexpression of CDCA3 occurs frequently during oral carcinogenesis and this overexpression might be associated closely with progression of OSCCs by preventing the arrest of cell-cycle progression at the G1 phase via decreased expression of the cyclin-dependent kinase inhibitors

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

    Science.gov (United States)

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

    2013-08-01

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

  2. Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.

    Science.gov (United States)

    Feillet, Céline; Krusche, Peter; Tamanini, Filippo; Janssens, Roel C; Downey, Mike J; Martin, Patrick; Teboul, Michèle; Saito, Shoko; Lévi, Francis A; Bretschneider, Till; van der Horst, Gijsbertus T J; Delaunay, Franck; Rand, David A

    2014-07-08

    Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

  3. Data for chromosome contacts and matched transcription profiles at three cell cycle phases in the fission yeast

    Directory of Open Access Journals (Sweden)

    Ralph S. Grand

    2015-06-01

    Full Text Available The data described in this article pertains to Grand et al. (2014, “Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure” [1]. Temperature sensitive Schizosaccharomyces pombe cell division cycle (cdc mutants, which are induced by a shift in temperature to 36 °C, were chosen for the analysis of genome structure in the G1 phase, G2 phase and mitotic anaphase of the cell cycle. Chromatin and total RNA were isolated from the same cell culture following synchronization. Two biological replicates were analyzed for each condition. The global, three-dimensional organization of the chromosomes was captured at high resolution using Genome Conformation Capture (GCC. GCC libraries and RNA samples were sequenced using an Illumina Hi-Seq 2000 platform (Beijing Genomics Institute (China. DNA sequences were processed using the Topography suite v1.19 [2] to obtain chromosome contact frequency matrices. RNA sequences were processed using the Cufflinks pipeline [3] to measure gene transcript levels and how these varied between the conditions. All sequence data, processed GCC and transcriptome files are available under the Gene Expression Omnibus (GEO accession number GSE52287 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52287.

  4. Concentration-dependent induction of reactive oxygen species, cell cycle arrest and apoptosis in human liver cells after nickel nanoparticles exposure.

    Science.gov (United States)

    Ahmad, Javed; Alhadlaq, Hisham A; Siddiqui, Maqsood A; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Ahamed, Maqusood

    2015-02-01

    Due to advent of nanotechnology, nickel nanoparticles (Ni NPs) are increasingly recognized for their utility in various applications including catalysts, sensors and electronics. However, the environmental and human health effects of Ni NPs have not been fully investigated. In this study, we examined toxic effects of Ni NPs in human liver (HepG2) cells. Ni NPs were prepared and characterized by X-ray diffraction, transmission electron microscopy and dynamic light scattering. We observed that Ni NPs (size, ∼28 nm; concentration range, 25-100 μg/mL) induced cytotoxicity in HepG2 cells and degree of induction was concentration-dependent. Ni NPs were also found to induce oxidative stress in dose-dependent manner evident by induction of reactive oxygen species and depletion of glutathione. Cell cycle analysis of cells treated with Ni NPs exhibited significant increase of apoptotic cell population in subG1 phase. Ni NPs also induced caspase-3 enzyme activity and apoptotic DNA fragmentation. Upregulation of cell cycle checkpoint gene p53 and bax/bcl-2 ratio with a concomitant loss in mitochondrial membrane potential suggested that Ni NPs induced apoptosis in HepG2 cells was mediated through mitochondrial pathway. This study warrants that applications of Ni NPs should be carefully assessed as to their toxicity to human health. © 2013 Wiley Periodicals, Inc.

  5. Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

    International Nuclear Information System (INIS)

    Meeteren, A. van; Wijk, R. van; Stap, J.; Deys, B.F.

    1984-01-01

    The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented. (author)

  6. Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

    Energy Technology Data Exchange (ETDEWEB)

    Meeteren, A van; Wijk, R van [Rijksuniversiteit Utrecht (Netherlands); Stap, J; Deys, B F [Amsterdam Univ. (Netherlands)

    1984-03-01

    The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented.

  7. Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid Emiliania huxleyi

    Science.gov (United States)

    Laguna, Richard; Romo, Jesus; Read, Betsy A.; Wahlund, Thomas M.

    2001-01-01

    Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga. PMID:11525973

  8. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    2007-12-31

    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  9. Deficient repair and degradation of DNA in X-irradiated L5178Y S/S cells: cell-cycle and temperature dependence

    International Nuclear Information System (INIS)

    Ueno, A.M.; Goldin, E.M.; Cox, A.B.; Lett, J.T.

    1979-01-01

    The rejoining of DNA strand breaks induced by X rays in the radiosensitive S/S variant of the L5178Y murine leukemic lymphoblast has been studied by alkaline-EDTA-sucrose sedimentation using swinging-bucket and zonal rotors. After irradiation, incubation resulted in an increase in DNA size, but the DNA structures were not restored in all cells, even when the x-ray dose was only 50 rad. Subsequently, 10 to 20 h after irradiation, heavily degraded DNA began to appear. When cells were irradiated at different parts of the cycle, the extent of DNA degradation varied in a fashion similar to survival: Least DNA degradation was found after irradiation at the most radioresistant stage (G 1 + 8 h), and most DNA degradation occurred after irradiation at the radiosensitive stage (G 1 ). Changes in cell survival caused by postirradiation hypothermia were also reflected in the extent of DNA degradation. Populations of G 1 cells, which show marked increases in survival after postirradiation hypothermic exposure, exhibited a lower level of DNA degradation, whereas populations of G 1 + 8 h cells, whose survival is affected little by postirradiation hypothermia, showed limited changes in DNA degradation. The onset of degradation was delayed by hypothermia in all cases

  10. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-01-01

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPARγ expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest

  11. Effects of low priming dose irradiation on cell cycle arrest of HepG2 cells caused by high dose irradiation

    International Nuclear Information System (INIS)

    Xia Jingguang; Jin Xiaodong; Chinese Academy of Sciences, Beijing; Li Wenjian; Wang Jufang; Guo Chuanling; Gao Qingxiang

    2005-01-01

    Human hepatoma cells hepG2 were irradiated twice by 60 Co γ-rays with a priming dose of 5 cGy and a higher dose of 3 Gy performed 4h or 8h after the low dose irradiation. Effects of the priming dose irradiation on cell cycle arrest caused by high dose were examined with flow cytometry. Cells in G 2 /M phase accumulated temporarily after the 5 cGy irradiation, and proliferation of tumor cells was promoted significantly by the low dose irradiation. After the 3 Gy irradiation, G 2 phase arrest occurred, and S phase delayed temporally. In comparison with 3 kGy irradiation only, the priming dose delivered 4h prior to the high dose irradiation facilitated accumulation of hepG2 cells in G 2 /M phase, whereas the priming dose delivered 8h prior to the high dose irradiation helped the cells to overcome G 2 arrest. It was concluded that effects of the priming dose treatment on cell cycle arrest caused by high dose irradiation were dependent on time interval between the two irradiations. (authors)

  12. Hericium erinaceus polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

    Science.gov (United States)

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

    2015-05-01

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

  13. Histone H1 interphase phosphorylation becomes largely established in G1 or early S phase and differs in G1 between T-lymphoblastoid cells and normal T cells

    Directory of Open Access Journals (Sweden)

    Gréen Anna

    2011-08-01

    Full Text Available Abstract Background Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G1, S and G2/M populations. Results We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G1 or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. Conclusion Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G1 may be affecting the G1/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.

  14. Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

    Directory of Open Access Journals (Sweden)

    Neel M Fofaria

    Full Text Available In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR and checkpoint kinase 1 (Chk1. Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb. Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.

  15. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

    Science.gov (United States)

    Escribano-Díaz, Cristina; Orthwein, Alexandre; Fradet-Turcotte, Amélie; Xing, Mengtan; Young, Jordan T F; Tkáč, Ján; Cook, Michael A; Rosebrock, Adam P; Munro, Meagan; Canny, Marella D; Xu, Dongyi; Durocher, Daniel

    2013-03-07

    DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Selective Cyclin-Dependent Kinase Inhibitors Discriminating between Cell Cycle and Transcriptional Kinases Future Reality or Utopia?

    Czech Academy of Sciences Publication Activity Database

    Wesierska-Gadek, J.; Kryštof, Vladimír

    2009-01-01

    Roč. 1171, - (2009), s. 228-241 ISSN 0077-8923 R&D Projects: GA ČR GA204/08/0511 Institutional research plan: CEZ:AV0Z50380511 Keywords : cell cycle * CYC202 * cyclin-dependent kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.670, year: 2009

  17. The p75NTR tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    International Nuclear Information System (INIS)

    Khwaja, Fatima; Tabassum, Arshia; Allen, Jeff; Djakiew, Daniel

    2006-01-01

    The p75 neurotrophin receptor (p75 NTR ) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75 NTR retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (ΔDD) dominant-negative antagonist of p75 NTR showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75 NTR -dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75 NTR expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75 NTR rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75 NTR was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75 NTR -dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75 NTR expressing prostate cancer cells

  18. Radiation-induced p53 protein response in the A549 cell line is culture growth-phase dependent

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, N.F.; Gurule, D.M.; Carpenter, T.R.

    1995-12-01

    One role of the p53 tumor suppressor protein has been recently revealed. Kastan, M.B. reported that p53 protein accumulates in cells exposed to ionizing radiation. The accumulation of p53 protein is in response to DNA damage, most importantly double-strand breaks, that results from exposure to ionizing radiation. The rise in cellular p53 levels is necessary for an arrest in the G{sub 1} phase of the cell cycle to provide additional time for DNA repair. The p53 response has also been demonstrated to enhance PCNA-dependent repair. p53 is thus an important regulator of the cellular response to DNA-damaging radiation. From this data, it can be concluded that the magnitude of the p53 response is not dependent on the phase of culture growth.

  19. Heterogenic final cell cycle by chicken retinal Lim1 horizontal progenitor cells leads to heteroploid cells with a remaining replicated genome.

    Directory of Open Access Journals (Sweden)

    Shahrzad Shirazi Fard

    Full Text Available Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.

  20. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    International Nuclear Information System (INIS)

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

    2006-01-01

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

  1. Studies on regulation of the cell cycle in fission yeast.

    Directory of Open Access Journals (Sweden)

    Miroslava Požgajová

    2015-05-01

    Full Text Available All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to understand multicellular organism we need to understand the life cycle of individual cells from which the organism comprises. The cell cycle is the life cycle of a single cell in the plant or animal body. It involves series of events in which components of the cell doubles and afterwards equally segregate into daughter cells. Such process ensures growth of the organism, and specialized reductional cell division which leads to production of gamets, assures sexual reproduction. Cell cycle is divided in the G1, S, G2 and M phase. Two gap-phases (G1 and G2 separate S phase (or synthesis and M phase which stays either for mitosis or meiosis. Essential for normal life progression and reproduction is correct chromosome segregation during mitosis and meiosis. Defects in the division program lead to aneuploidy, which in turn leads to birth defects, miscarriages or cancer. Even thou, researchers invented much about the regulation of the cell cycle, there is still long way to understand the complexity of the regulatory machineries that ensure proper segregation of chromosomes. In this paper we would like to describe techniques and materials we use for our studies on chromosome segregation in the model organism Schizosaccharomyces pombe.

  2. Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

    Science.gov (United States)

    Panzarini, Elisa; Mariano, Stefania; Vergallo, Cristian; Carata, Elisabetta; Fimia, Gian Maria; Mura, Francesco; Rossi, Marco; Vergaro, Viviana; Ciccarella, Giuseppe; Corazzari, Marco; Dini, Luciana

    2017-06-01

    This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10 3 or 2×10 4 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag + release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10 4 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag + release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Cell cycle variation in x-ray survival for cells from spheroids measured by volume cell sorting

    International Nuclear Information System (INIS)

    Freyer, J.P.; Wilder, M.E.; Raju, M.R.

    1984-01-01

    Considerable work has been done studying the variation in cell survival as a function of cell cycle position for monolayers or single cells exposed to radiation. Little is known about the effects of multicellular growth on the relative radiation sensitivity of cells in different cell cycle stages. The authors have developed a new technique for measuring the response of cells, using volume cell sorting, which is rapid, non-toxic, and does not require cell synchronization. By combining this technique with selective spheroid dissociation,they have measured the age response of cells located at various depths in EMT6 and Colon 26 spheroids. Although cells in the inner region had mostly G1-phase DNA contents, 15-20% had S- and G2-phase DNA contents. Analysis of these cells using BrdU labeling and flow cytometric analysis with a monoclonal antibody to BrdU indicated that the inner region cells were not synthesizing DNA. Thus, the authors were able to measure the radiation response of cells arrested in G1, S and G2 cell cycle phases. Comparison of inner and outer spheroid regions, and monolayer cultures, indicates that it is improper to extrapolate age response data in standard culture conditions to the situation in spheroids

  4. Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci)

    Energy Technology Data Exchange (ETDEWEB)

    Honda-Uezono, Asumi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Kaida, Atsushi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Michi, Yasuyuki; Harada, Kiyoshi [Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Yoshiki; Hayashi, Yoshio [Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392 (Japan); Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Fucci visualizes cell cycle by green and red fluorescence. Black-Right-Pointing-Pointer Plinabulin, induced unusual red fluorescence at M-phase in HeLa-Fucci cells. Black-Right-Pointing-Pointer The unusual pattern was followed by mitotic catastrophe. Black-Right-Pointing-Pointer The unusual pattern may be an early indicator of cell death in HeLa cells. -- Abstract: Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

  5. Centrosome/Cell cycle uncoupling and elimination in the endoreduplicating intestinal cells of C. elegans.

    Directory of Open Access Journals (Sweden)

    Yu Lu

    Full Text Available The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

  6. S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

    Directory of Open Access Journals (Sweden)

    AGM Mostofa

    2018-04-01

    Full Text Available Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  10. Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

    International Nuclear Information System (INIS)

    Jung, T.

    2000-01-01

    The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.) [de

  11. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

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

    Science.gov (United States)

    Fleisig, Helen; Wong, Judy

    2012-05-22

    metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

  13. CHANGES OF BUOYANT DENSITY DURING THE S-PHASE OF THE CELL-CYCLE - DIRECT EVIDENCE DEMONSTRATED IN ACUTE MYELOID-LEUKEMIA BY FLOW-CYTOMETRIC

    NARCIS (Netherlands)

    DAENEN, S; HUIGES, W; MODDERMAN, E; HALIE, MR

    Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows

  14. [Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

    Science.gov (United States)

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

    2017-12-01

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

  15. Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.

    Science.gov (United States)

    Senese, S; Lo, Y C; Huang, D; Zangle, T A; Gholkar, A A; Robert, L; Homet, B; Ribas, A; Summers, M K; Teitell, M A; Damoiseaux, R; Torres, J Z

    2014-10-16

    Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF(V600E) melanomas.

  16. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Directory of Open Access Journals (Sweden)

    Ludmila Alekseeva

    Full Text Available Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  17. Development of cell-cycle checkpoint therapy for solid tumors.

    Science.gov (United States)

    Tamura, Kenji

    2015-12-01

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

  18. DNA double-strand break and apoptosis induction in human lymphocytes in different cycle cell phases by 60Co gamma rays and Bragg peak protons of a medical beam

    International Nuclear Information System (INIS)

    Khachenkova, A.A.; Boreyko, A.V.; Mozhaeva, A.V.; Chausov, V.N.; Ravnachka, I.I.; Amov, I.; Tiunchik, S.I.

    2009-01-01

    A comparative analysis is made of the regularities in the formation of DNA double-strand break and apoptosis induction in peripheral human blood lymphocytes in different cell cycle phases after 60 Co gamma and extended Bragg peak proton irradiation. It is shown that the formation of apoptotic cells in a lymphocyte population increases linearly in all the cell cycle stages after proton irradiation. The maximal DNA double-strand break and apoptosis yield in lymphocytes is observed in the S phase of the cell cycle

  19. Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

    Science.gov (United States)

    Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

    2011-04-01

    The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  20. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

    Science.gov (United States)

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R; Scherer, Norbert F

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS).

  1. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    Energy Technology Data Exchange (ETDEWEB)

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  2. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    Science.gov (United States)

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  3. Clastogenic adaption of Vicia faba root tip meristem cells after consecutive treatments with S-phase dependent and S-phase independent agents

    International Nuclear Information System (INIS)

    Heindorff, K.; Schubert, I.; Rieger, R.; Michaelis, A.

    1987-01-01

    Pretreatment of Vicia faba root tip meristems with low doses of S-phase independent clastogens, such as X-rays or bleomycin, prior to a high (challenge) dose of S-phase dependent clastogens, such as alkylating agents (TEM, Trenimon) or the pyridazine derivative MH, led to decreased challenge treatment-induced aberration frequencies, i.e., clastogenic adaptation. Using the inverse treatment sequence bleomycin and MH proved to be able to substitute for each other in provoking clastogenic (cross) adaptation while bleomycin and alkylating agents were unable to do so. The data support the assumption of inducible cellular functions that become triggered by low clastogen doses and additionally describe some particular properties of bleomycin when used for conditioning. Bleomycin proved to be capable to exert protection independent of the agent used for challenge treatment. (author)

  4. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1......) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  5. Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    Full Text Available Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

  6. Dihydromyricetin induces cell cycle arrest and apoptosis in melanoma SK-MEL-28 cells.

    Science.gov (United States)

    Zeng, Guofang; Liu, Jie; Chen, Hege; Liu, Bin; Zhang, Qingyu; Li, Mingyi; Zhu, Runzhi

    2014-06-01

    Dihydromyricetin (DHM) exhibits multiple pharmacological activities; however, the role of DHM in anti-melanoma activities and the underlying molecular mechanisms are unclear. The aim of the present study was to evaluate the effects of DHM on cell proliferation, cell cycle distribution and apoptosis in the human melanoma SK-MEL-28 cell line, and to explore the related mechanisms. The effect of DHM on cell proliferation was investigated by MTT assay, and cell cycle distribution was determined by flow cytometry. TUNEL assay was used to evaluate DHM-mediated apoptosis, and western blotting was applied to examine expression levels of p53, p21, Cdc25A, Cdc2, P-Cdc2, Bax, IKK-α, NF-κB p65, p38 and P-p38 proteins. The results revealed that DHM suppressed cell proliferation of SK-MEL-28 cells in a concentration- and time-dependent manner, and caused cell cycle arrest at the G1/S phase. DHM increased the production of p53 and p21 proteins and downregulated the production of Cdc25A, Cdc2 and P-Cdc2 proteins, which induced cell cycle arrest. Additionally, DHM significantly induced the apoptosis of SK-MEL-28 cells, and enhanced the expression levels of Bax proteins and decreased the protein levels of IKK-α, NF-κB (p65) and P-p38. The results suggest that DHM may be a novel and effective candidate agent to inhibit the growth of melanoma.

  7. Neurosecretory cells of the amygdaloid complex during estrous cycle.

    Science.gov (United States)

    Akhmadeev, A V; Kalimullina, L B

    2005-02-01

    Ultrastructure of neurosecretory cells of the dorsomedial nucleus of the cerebral amygdaloid complex (one of the main zones of sexual dimorphism) was studied in different phases of the estrous cycle. The characteristics of the "light" and "dark" cells change depending on the concentrations of sex steroids during estrus and metestrus.

  8. The p75{sup NTR} tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Khwaja, Fatima [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States); Tabassum, Arshia [Toronto Western Hospital, Toronto, ON, M5T258 (Canada); Allen, Jeff [National Center for Complementary and Alternative Medicine, N.I.H., Bethesda, MD 20892 (United States); Djakiew, Daniel [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States) and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057-1436 (United States)

    2006-03-24

    The p75 neurotrophin receptor (p75{sup NTR}) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75{sup NTR} retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted ({delta}DD) dominant-negative antagonist of p75{sup NTR} showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75{sup NTR}-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75{sup NTR} expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75{sup NTR} rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75{sup NTR} was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75{sup NTR}-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75{sup NTR} expressing prostate cancer cells.

  9. PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei [Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China); Zhu, Jiang [Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China); Ozaki, Toshinori [Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuohku, Chiba 260-8717 (Japan); Bu, Youquan, E-mail: buyqcn@aliyun.com [Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China)

    2015-03-13

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

  10. PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC)

    International Nuclear Information System (INIS)

    Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei; Zhu, Jiang; Ozaki, Toshinori; Bu, Youquan

    2015-01-01

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

  11. Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

    Science.gov (United States)

    Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

    2018-03-02

    Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

  12. An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

    Science.gov (United States)

    Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

    2018-01-01

    Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving

  13. Tangeretin and its metabolite 4′-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K

    Science.gov (United States)

    Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, MR

    2011-01-01

    BACKGROUND AND PURPOSE The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4′-hydroxy-5,6,7,8-tetramethoxyflavone (4′-OH-TMF) in this effect. EXPERIMENTAL APPROACH We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [3H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. KEY RESULTS Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4′-OH-TMF. Low micromolar concentrations of tangeretin or 4′-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4′-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. CONCLUSIONS AND IMPLICATIONS Tangeretin and 4′-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4′-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. PMID:21198542

  14. Study of the cell cycle control for human malignant mesothelioma lines. Interferon and radiations effect; Etude de la regulation du cycle cellulaire de lignees de mesotheliome malin humain. Effet de l'interferon et des radiations

    Energy Technology Data Exchange (ETDEWEB)

    Vivo, C

    1999-07-01

    In order to better understand the inhibition mechanisms of the IFN-R-HU on tumoral development, the IFN-R-U effect on MM lines has been studied. Three groups of lines has been distinguished: eight sensitive lines, two intermediate and three resistant. The sensitive lines showed a triple locking of the cell cycle: in phases S, G1 and G2. The study of the cell cycle control points function, realized by the MM lines radiation exposure showed the points function on G1/S and-or on G2/M and the dependence or non dependence of the cycle stop of the protein P53 and P21 W at F1/CIP1. (A.L.B.)

  15. Cell cycle delays in synchronized cell populations following irradiation with heavy ions

    International Nuclear Information System (INIS)

    Scholz, M.

    1992-11-01

    Mammalian cells subjected to irradiation with heavy ions were investigated for cell cycle delays. The ions used for this purpose included Ne ions in the LET range of 400 keV/μm just as well as uranium ions of 16225 keV/μm. The qualitative changes in cell cycle progression seen after irradiation with Ne ions (400 keV/μm) were similar to those observed in connection with X-rays. Following irradiation with extremely heavy ions (lead, uranium) the majority of cells were even at 45 hours still found to be in the S phase or G 2 M phase of the first cycle. The delay cross section 'σ-delay' was introduced as a quantity that would permit quantitative comparisons to be carried out between the changes in cell progression and other effects of radiation. In order to evaluate the influence of the number of hits on the radiation effect observed, the size of the cell nucleus was precisely determined with reference to the cycle phase and local cell density. A model to simulate those delay effects was designed in such a way that account is taken of this probability of hit and that the results can be extrapolated from the delay effects after X-irradiation. On the basis of the various probabilities of hit for cells at different cycle stages a model was developed to ascertain the intensified effect following fractionated irradiation with heavy ions. (orig./MG) [de

  16. Rates of incorporation of radioactive molecules during the cell cycle

    International Nuclear Information System (INIS)

    Gray, J.W.; Pallavicini, M.G.; George, Y.S.; Groppi, V.; Look, M.; Dean, P.N.

    1981-01-01

    We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine ( 3 H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3 H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) 3 H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing 3 H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of 3 H-TdR and 35 S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3 H-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3 H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of 35 S-methionine increased continuously with cell size and DNA content. Incorporation of 3 H-TdR in CHO cells was proportional to DNA synthesis

  17. 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).

    Science.gov (United States)

    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.

  18. TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase

    International Nuclear Information System (INIS)

    Takeuchi, Shin; Kasamatsu, Atsushi; Yamatoji, Masanobu; Nakashima, Dai; Endo-Sakamoto, Yosuke; Koide, Nao; Takahara, Toshikazu; Shimizu, Toshihiro; Iyoda, Manabu; Ogawara, Katsunori; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

    2017-01-01

    TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs. - Highlights: • TEAD4 contributes to tumor progression in OSCCs. • TEAD4 knockdown results in cell-cycle arrest at the G1phase in OSCC cells. • In TEAD4 knockdown cells, the amount of YAP in the nucleus decreases. • Activation of the TEAD4-YAP complex is an important factor in OSCC tumor growth. • TEAD4 might be a critical biomarker and a therapeutic target for OSCCs.

  19. Therapeutic Cell-Cycle-Decoy Efficacy of a Telomerase-Dependent Adenovirus in an Orthotopic Model of Chemotherapy-Resistant Human Stomach Carcinomatosis Peritonitis Visualized With FUCCI Imaging.

    Science.gov (United States)

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

    2017-11-01

    We have established an orthotopic nude-mouse model of gastric cancer carcinomatosis peritonitis, a recalcitrant disease in human patients. Human MKN45 poorly-differentiated human gastric cancer cells developed carcinomatosis peritonitis upon orthotopic transplantation in nude mice. The MKN45 cells expressed the fluorescent ubiquitination-based cell cycle indicator (FUCCI) that color codes the phases of the cell cycle. The intra-peritoneal tumors and ascites contained mostly quiescent G 1 /G o cancer cells visualized as red by FUCCI imaging. Cisplatinum (CDDP) treatment did not reduce bloody ascites, and larger tumors formed in the peritoneal cavity after CDDP treatment in an early-stage carcinomatosis peritonitis orthotopic mouse model. Paclitaxel-treated mice had reduced ascites, but also had large tumor masses in the peritonium after treatment with cancer cells mostly in G 0 /G 1 , visualized by FUCCI red. In contrast, OBP-301 telomerase-dependent adenovirus-treated mice had no ascites and only small tumor nodules consisting of cancer cells mostly in S/G 2 phases in the early-stage carcinomatosis peritonitis model, visualized by FUCCI green. Furthermore, OBP-301 significantly reduced the size of tumors (P < 0.01) and ascites even in a late-stage carcinomatosis peritonitis model. These results suggest that quiescent peritoneally-disseminated gastric cancer cells are resistant to conventional chemotherapy, but OBP-301 significantly reduced the weight of the tumors and increased survival, suggesting clinical potential. J. Cell. Biochem. 118: 3635-3642, 2017. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

    International Nuclear Information System (INIS)

    Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing; Guo Chun; Zhu Faliang; Yang Qiaozi; Gao Guimin; Gong Yaoqin; Shao Changshun

    2009-01-01

    Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis

  1. Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Guo Chun; Zhu Faliang [Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Yang Qiaozi [Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States); Gao Guimin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Gong Yaoqin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China)], E-mail: yxg8@sdu.edu.cn; Shao Changshun [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)], E-mail: shao@biology.rutgers.edu

    2009-03-09

    Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis.

  2. Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

    Directory of Open Access Journals (Sweden)

    Giddings Ian

    2011-06-01

    Full Text Available Abstract Background Benzo[a]pyrene (BaP is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP 1A1 and CYP1B1, which are involved in BaP metabolism. Results Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE, the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. Conclusions This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell

  3. Radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) within the cell cycle in vitro

    International Nuclear Information System (INIS)

    Latz, Detlev; Fleckenstein, Katharina; Eble, Michael; Blatter, Johannes; Wannenmacher, Michael; Weber, Klaus J.

    1998-01-01

    Purpose: Gemcitabine (2',2'-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle-specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells. Methods and Materials: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle-specific enhancement ratios (ER) were calculated from the survival curve parameters. Results: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)]. Conclusions: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been

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

    Directory of Open Access Journals (Sweden)

    Choi Hyun

    2009-05-01

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

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

    Science.gov (United States)

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

    2009-05-29

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

  6. Outcome of Treatment of Human HeLa Cervical Cancer Cells With Roscovitine Strongly Depends on the Dosage and Cell Cycle Status Prior to the Treatment

    Czech Academy of Sciences Publication Activity Database

    Wesierska-Gadek, J.; Borza, A.; Walzi, E.; Kryštof, Vladimír; Maurer, M.; Komina, O.; Wandl, S.

    2009-01-01

    Roč. 106, č. 5 (2009), s. 937-955 ISSN 0730-2312 Institutional research plan: CEZ:AV0Z50380511 Keywords : APOPTOSIS * CELL CYCLE ARREST * CYCLIN-DEPENDENT KINASES Subject RIV: ED - Physiology Impact factor: 2.935, year: 2009

  7. Cancer cells mimic in vivo spatial-temporal cell-cycle phase distribution and chemosensitivity in 3-dimensional Gelfoam® histoculture but not 2-dimensional culture as visualized with real-time FUCCI imaging.

    Science.gov (United States)

    Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Uehara, Fuminaru; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Zhao, Ming; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2015-01-01

    The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We previously reported monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor, intravitally in live mice, using a fluorescence ubiquitination-based cell-cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after cessation of chemotherapy. These results suggested why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. In the present report, we used FUCCI imaging and Gelfoam® collagen-sponge-gel histoculture, to demonstrate in real time, that the cell-cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is highly similar, whereby only the surface cells proliferate and interior cells are quiescent in G0/G1. This is in contrast to 2D culture where most cancer cells cycle. Similarly, the cancer cells responded similarly to toxic chemotherapy in Gelfoam® culture as in vivo, and very differently than cancer cells in 2D culture which were much more chemosensitive. Gelfoam® culture of FUCCI-expressing cancer cells offers the opportunity to image the cell cycle of cancer cells continuously and to screen for novel effective therapies to target quiescent cells, which are the majority in a tumor and which would have a strong probability to be effective in vivo.

  8. Labeling of lectin receptors during the cell cycle.

    Science.gov (United States)

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  9. The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

    Science.gov (United States)

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

    2014-01-01

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

  10. Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis

    International Nuclear Information System (INIS)

    Park, Woo Hyun; Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo

    2007-01-01

    Pyrogallol as a catechin compound has been employed as an O 2 · - generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC 50 of about 60 μM for 48 h using MTT assay. Pyrogallol (100 μM) did not alter intracellular H 2 O 2 level and catalase activity, but increased the intracellular O 2 · - level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 μM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (ΔΨ m ), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells

  11. Methods for Synchronization and Analysis of the Budding Yeast Cell Cycle.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    Like other eukaryotes, budding yeast temporally separate cell growth and division. DNA synthesis is distinct from chromosome segregation. Storage carbohydrates are accumulated slowly and then rapidly liquidated once per cycle. Cyclin-dependent kinase associates with multiple different transcriptionally and posttranslationally regulated cyclins to drive the cell cycle. These and other crucial events of cellular growth and division are limited to narrow windows of the cell cycle. Many experiments in the yeast laboratory treat a culture of cells as a homogeneous mixture. Measurements of asynchronous cultures are, however, confounded by the presence of cells in various cell cycle stages; measuring a population average in unsynchronized cells provides at best a decreased signal and at worst an artifactual result. A number of experimentally tractable methods have been developed to generate populations of yeast cells that are synchronized with respect to cell cycle phase. Robust methods for determining cell cycle position have also been developed. These methods are introduced here. © 2017 Cold Spring Harbor Laboratory Press.

  12. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Science.gov (United States)

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-07-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  13. Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation

    International Nuclear Information System (INIS)

    Willner, Jochen; Vordermark, Dirk; Schmidt, Michael; Gassel, Andreamaria; Flentje, Michael; Wirtz, Hubert

    2003-01-01

    Purpose: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo. Methods and Materials: Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using 3 H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index. Results: During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G 2 /M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls. Conclusion: In contrast to literature data, we observed no direct

  14. Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

    International Nuclear Information System (INIS)

    Tokita, N.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

  15. Effects of caffeine on X-irradiated synchronous, asynchronous and plateau phase mouse ascites cells: the importance of progression through the cell cycle for caffeine enhancement of killing

    International Nuclear Information System (INIS)

    Iliakis, G.; Nuesse, M.

    1983-01-01

    Caffeine potentiated the killing effect of X-rays on exponentially growing cells giving rise to exponential curves (D 0 =(0.8+-0.05)Gy) at 4mM and 14 hours treatment. Irradiated plateau phase cells were less sensitive. Exponentially growing cells also became less sensitive to the effects of caffeine when they were incubated in the conditioned medium of plateau phase cells(C-medium) in which cell growth was considerably inhibited. Low caffeine concentrations(2mM) enhanced X-ray induced killing of cells irradiated in G 1 -,G 1 /S- or S-phase, but more effectively G 2 -phase cells. High caffeine concentrations (6mM) enhanced killing of cells in all phases of the cell cycle. Incubation of synchronized populations in C-medium during treatment with caffeine (2mM and 6mM) resulted in less potentiation than in cells treated in fresh medium. The expression of X-ray induced potentially lethal damage caused by 6mM caffeine in cells irradiated in various phases resulted in an exponential survival curve with a mean lethal dose of (0.8+-0.05)Gy, but the time of caffeine treatment necessary to reach this curve was different for cells irradiated in different phases. PLD repair, measured as loss of sensitivity to 6mM caffeine (4 hours treatment) was of 1-2 hours duration. (author)

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

    Science.gov (United States)

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

    2011-10-01

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

  17. Prognostic value of lymphoma-specific S-phase fraction compared with that of other cell proliferation markers

    Energy Technology Data Exchange (ETDEWEB)

    Holte, H.; Kvaloey, S. [Dept. of Oncology, Univ. of Oslo (Norway); Suo Zhenhe; Langholm, R. [Dept. of Pathology, Univ. of Oslo (Norway); Smeland, E.B. [Dept. of Immunology, Univ. of Oslo (Norway); Stokke, T. [Dept. of Biophysics, Univ. of Oslo (Norway)

    1999-11-01

    The proliferation-associated antigens Ki67 (immunohistochemistry) and proliferative cell nuclear antigen (PCNA) (immunohistochemistry and immunoblotting) were analysed together with DNA synthesis ({sup 3}H-thymidine incorporation) and cell-cycle distribution (tumour-specific S-phase fraction determined by flow cytometry) in lymph node suspensions from 63 patients with newly diagnosed B-Cell non-Hodgkin`s lymphomas. Details of clinical parameters, treatment and patient outcome were available for all patients, and retrospectively analysed. Of the proliferation-associated parameters, only high S-phase fraction (p < 0.00001) and high PCNA expression by immunoblotting (p=0.012) were predictive of a poor prognosis. Of the conventional parameters, high-grade malignancy, high International Prognostic Index (IPI) score, bulky disease and presence of B symptoms predicted a patient for poor survival. High S-phase fraction was predictive of a short survival for the low-grade lymphomas analyses separately (p < 0.00001), as well as for patients treated with an Adriamycin- and a non-Adriamycin-containing regimen (p < 0.005 for both groups). In a multivariate analysis, S-phase fraction (p=0.00006), IPI score (p=0.015) and B symptoms (p=0.017) had independent prognostic values, but not histological grade. (orig.)

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

    Science.gov (United States)

    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.

  19. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2001-01-01

    Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified...

  20. Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression.

    Directory of Open Access Journals (Sweden)

    Mathieu Dalvai

    Full Text Available Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7 the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

  1. Differential S-phase progression after irradiation of p53 functional versus non-functional tumour cells

    Directory of Open Access Journals (Sweden)

    Zölzer Friedo

    2014-12-01

    Full Text Available Background. Many pathways seem to be involved in the regulation of the intra-S-phase checkpoint after exposure to ionizing radiation, but the role of p53 has proven to be rather elusive. Here we have a closer look at the progression of irradiated cells through S-phase in dependence of their p53 status.

  2. Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs

    Directory of Open Access Journals (Sweden)

    Mei-Yin Chang

    2015-11-01

    Full Text Available Plantaginaceae, a popular traditional Chinese medicine, has long been used for treating various diseases from common cold to cancer. Linalool is one of the biologically active compounds that can be isolated from Plantaginaceae. Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible tumor cells. However, the signaling pathway for apoptosis remains undefined. In this study, the cytotoxic effect of linalool on human cancer cell lines was investigated. Water-soluble tetrazolium salts (WST-1 based colorimetric cellular cytotoxicity assay, was used to test the cytotoxic ability of linalool against U937 and HeLa cells, and flow cytometry (FCM and genechip analysis were used to investigate the possible mechanism of apoptosis. These results demonstrated that linalool exhibited a good cytotoxic effect on U937 and HeLa cells, with the IC50 value of 2.59 and 11.02 μM, respectively, compared with 5-FU with values of 4.86 and 12.31 μM, respectively. After treating U937 cells with linalool for 6 h, we found an increased sub-G1 peak and a dose-dependent phenomenon, whereby these cells were arrested at the G0/G1 phase. Furthermore, by using genechip analysis, we observed that linalool can promote p53, p21, p27, p16, and p18 gene expression. Therefore, this study verified that linalool can arrest the cell cycle of U937 cells at the G0/G1 phase and can arrest the cell cycle of HeLa cells at the G2/M phase. Its mechanism facilitates the expression of the cyclin-dependent kinases inhibitors (CDKIs p53, p21, p27, p16, and p18, as well as the non-expression of cyclin-dependent kinases (CDKs activity.

  3. [EEG alpha indices in dependence on the menstrual cycle phase and salivary progesterone].

    Science.gov (United States)

    Bazanova, O M; Kondratenko, A V; Kuz'minova, O I; Muravleva, K B; Petrova, S E

    2014-01-01

    The effects of the neurohumoral status on the EEG alpha - activity indices were studied in a within-subject design with 78 women aged 18-27 years during 1-2 menstrual cycle. Psychometric and EEG indices of alpha waves basal body temperature, saliva progesterone and cortisol level were monitored every 2-3 days. Menstrual and follicular recording sessions occurred before the ovulatory temperature rise, luteal recording session--after increasing progesterone level more than 20% respect to previous day and premenstrual sessions after decreasing progesterone level more that 20% respect to previous day. The design consisted of rest and task periods EEG, EMG and ECG recordings. Half the subjects began during their menstrual phase and half began during their luteal phase. All 5 phases were compared for differences between psychometric features EEG alpha activity, EMG and ECG baseline resting levels, as well as for reactivity to cognitive task. The results showed menstrual phase differences in all psychometric and alpha EEG indices. The cognitive fluency, alpha peak frequency, alpha band width, power in alpha-2 frequency range are maximal at luteal, alpha visual activation and reactivity to cognitive task performance--at follicular phase. The hypothesis that the EEG alpha activity depends on the hormonal status supported by the positive association salivary progesterone level with the alpha peak frequency, power in the alpha-2 band and negative--with the power of the alpha-1 band. According these results, we conclude that psycho-physiological recording sessions with women might be provided with a glance to phase of menstrual cycle.

  4. Induction of apoptosis and antiproliferative activity of naringenin in human epidermoid carcinoma cell through ROS generation and cell cycle arrest.

    Directory of Open Access Journals (Sweden)

    Md Sultan Ahamad

    Full Text Available A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01 with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001 dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation.

  5. Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

    Directory of Open Access Journals (Sweden)

    Li HF

    2015-06-01

    Full Text Available Huai-Feng Li,1–3,* Xu-An Wang,1–3,* Shan-Shan Xiang,1–3,* Yun-Ping Hu,1–3 Lin Jiang,1–3 Yi-Jun Shu,1–3 Mao-Lan Li,1–3 Xiang-Song Wu,1–3 Fei Zhang,1–3 Yuan-Yuan Ye,1–3 Hao Weng,1–3 Run-Fa Bao,1–3 Yang Cao,1–3 Wei Lu,1–3 Qian Dong,1–3 Ying-Bin Liu1–3 1Department of General Surgery, 2Laboratory of General Surgery, 3Institute of Biliary Tract Disease, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Oleanolic acid (OA, a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma. Keywords: oleanolic acid, gallbladder carcinoma, apoptosis, cell cycle arrest, mitochondrial pathway

  6. Distinct mechanisms act in concert to mediate cell cycle arrest.

    Science.gov (United States)

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

    2009-01-20

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

  7. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  8. A retinoblastoma orthologue is a major regulator of S-phase, mitotic, and developmental gene expression in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Kimchi Strasser

    Full Text Available The retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression thus stabilizing cells in G1. Second, Rb interacts with factors that induce cell-cycle exit and terminal differentiation. Dictyostelium lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, rblA.Using microarray analysis and mRNA-Seq transcriptional profiling, we show that RblA strongly represses genes whose products are involved in S phase and mitosis. Both S-phase and mitotic genes are upregulated at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation.Like its mammalian counterpart Dictyostelium, RblA plays a dual role, regulating cell-cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes.

  9. Prognostic value of lymphoma-specific S-phase fraction compared with that of other cell proliferation markers

    International Nuclear Information System (INIS)

    Holte, H.; Kvaloey, S.; Suo Zhenhe; Langholm, R.; Smeland, E.B.; Stokke, T.

    1999-01-01

    The proliferation-associated antigens Ki67 (immunohistochemistry) and proliferative cell nuclear antigen (PCNA) (immunohistochemistry and immunoblotting) were analysed together with DNA synthesis ( 3 H-thymidine incorporation) and cell-cycle distribution (tumour-specific S-phase fraction determined by flow cytometry) in lymph node suspensions from 63 patients with newly diagnosed B-Cell non-Hodgkin's lymphomas. Details of clinical parameters, treatment and patient outcome were available for all patients, and retrospectively analysed. Of the proliferation-associated parameters, only high S-phase fraction (p < 0.00001) and high PCNA expression by immunoblotting (p=0.012) were predictive of a poor prognosis. Of the conventional parameters, high-grade malignancy, high International Prognostic Index (IPI) score, bulky disease and presence of B symptoms predicted a patient for poor survival. High S-phase fraction was predictive of a short survival for the low-grade lymphomas analyses separately (p < 0.00001), as well as for patients treated with an Adriamycin- and a non-Adriamycin-containing regimen (p < 0.005 for both groups). In a multivariate analysis, S-phase fraction (p=0.00006), IPI score (p=0.015) and B symptoms (p=0.017) had independent prognostic values, but not histological grade. (orig.)

  10. Proliferation marker pKi-67 affects the cell cycle in a self-regulated manner.

    Science.gov (United States)

    Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Duchrow, Michael

    2002-01-01

    The proliferation marker pKi-67 is commonly used in research and pathology to detect proliferating cells. In a previous work, we found the protein to be associated with regulators of the cell cycle, controlling S-phase progression, as well as entry into and exit from mitosis. Here we investigate whether pKi-67 has a regulative effect on the cell cycle itself. For that purpose we cloned four fragments of pKi-67, together representing nearly the whole protein, and an N-terminal pKi-67 antisense oligonucleotide into a tetracycline inducible gene expression system. The sense fragments were C-terminally modified by addition of either a nuclear localization sequence (NLS) or a STOP codon to address the impact of their intracellular distribution. FACS based cell cycle analysis revealed that expression of nearly all pKi-67 domains and the antisense oligonucleotide led to a decreased amount of cells in S-phase and an increased number of cells in G(2)/M- and G(1)-phase. Subsequent analysis of the endogenous pKi-67 mRNA and protein levels revealed that the constructs with the most significant impact on the cell cycle were able to silence pKi-67 transcription as well. We conclude from the data that pKi-67 influences progression of S-phase and mitosis in a self-regulated manner and, therefore, effects the cell cycle checkpoints within both phases. Furthermore, we found pKi-67 mediates an anti-apoptotic effect on the cell and we verified that this marker, although it is a potential ribosomal catalyst, is not expressed in differentiated tissues with a high transcriptional activity. Copyright 2002 Wiley-Liss, Inc.

  11. SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yuanyuan; He, Haiyun [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Peng, Feng [Department of Thoracic Oncology of the Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Zhao, Yinglan, E-mail: alancenxb@sina.com [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

  12. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva

    2005-07-01

    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  13. Enhancement of postreplication repair in ultraviolet-light-irradiated Chinese hamster cells by irradiation in G2 or s-phase

    International Nuclear Information System (INIS)

    D'Ambrosio, S.M.; Aebersold, P.M.; Setlow, R.B.

    1978-01-01

    Postreplication repair in synchronous Chinese hamster cells was determined after split doses of ultraviolet (uv) radiation. Repair was enhanced by irradiation of cells in G 2 or S-phase with a small dose of uv radiation at least 1.5 h before a three-fold larger dose of uv. There was significantly greater enhancement when the first dose was given in G 2 than when it was given in the S-phase 0.5 to 1.5 h before the test dose. These data indicate that enhancement of postreplication repair does not require active DNA replication and qualitatively is independent of when in the cell cycle the cells are irradiated

  14. A cell cycle role for the epigenetic factor CTCF-L/BORIS.

    Directory of Open Access Journals (Sweden)

    Manuel Rosa-Garrido

    Full Text Available CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

  15. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    International Nuclear Information System (INIS)

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-01-01

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence

  16. ROLE OF PI3K-AKT-mTOR AND Wnt SIGNALING PATHWAYS IN G1-S TRANSITION OF CELL CYCLE IN CANCER CELLS

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    LAKSHMIPATHI eVADLAKONDA

    2013-04-01

    Full Text Available The PI3K–Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR is a highly deregulated pathway in cancers. There is a reciprocal relation between the Akt phosphorylation and mTOR complexes. Akt phosphorylated at T308 activates mTORC1 by inhibition of the tuberous sclerosis complex (TSC1/2, where as mTORC2 is recognized as the kinase that phosphorylates Akt at S473. Recent developments in the research on regulatory mechanisms of autophagy places mTORC1 mediated inhibition of autophagy at the central position in activation of proliferation and survival pathways in cells. Autophagy is a negative regulator of Wnt signaling pathway and the downstream effectors of Wnt signaling pathway, cyclin D1 and the c-Myc, are the key players in initiation of cell cycle and regulation of the G1-S transition in cancer cells. Production of reaction oxygen species (ROS, a common feature of a cancer cell metabolism, activates several downstream targets like the transcription factors FoxO, which play key roles in promoting the progression of cell cycle. A model is presented on the role of PI3K -Akt - mTOR and Wnt pathways in regulation of the progression of cell cycle through Go-G1-and S phases.

  17. The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

    Science.gov (United States)

    Mizejewski, G J

    2016-09-01

    The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

  18. Cell-cycle-dependent repair of heavy-ion damage

    International Nuclear Information System (INIS)

    Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

    1985-01-01

    Synchronized human T-1 cells have been used to investigate the G1-phase age dependence of repair of potentially lethal damage (PLDR). The cells were irradiated with single doses of either 225 kVp X rays or Bragg-peak 425 MeV/μ neon ions at ages between 1.5 and 6.0 hrs after mitotic selection, and then either trypsinized and plated immediately, or held at 37 0 C for 6 hrs in PBS, or PBS containing 60μM of the DNA-polymerase-inhibitor 1-β-D-arabinofurano-syladenine (β-araA) before trypsinization and plating. Delayed plating showed significant PLDR at all ages irradiated with X rays, with the increase of survival varying between 2- to 8-fold. At equivalent survival levels, there was a reduced capacity for PLDT at each cell age irradiated with neon ions. In early G1 after neon-ion exposures, delayed plating actually enhanced cell killing; whereas, in late G1 the survival increased about 2-fold. β-araA almost completely eliminated the PLDR after X rays, reducing the survival to that measured with immediate plating. β-araA slightly enhanced neon-ion cell killing at all cell ages

  19. Effect of low dose radiation on cell cycle and expression of its related proteins of HCT-8 cells

    International Nuclear Information System (INIS)

    Xu Ying; Ma Kewei; Li Wei; Wang Guanjun

    2009-01-01

    Objective: To study the effects of low dose radiation (LDR) on cell cycle and the expression of its related proteins of HCT-8 cells and provide theoretical basis for clinical application of LDR. Methods: Human colon carcinoma cells (HCT-8) cultivated in vitro were divided into seven groups: sham radiation group (0 mGy), LDR groups (25, 50, 75, 100 and 200 mGy) and high dose radiation group (1000 mGy). The proliferation rate was detected with the method of cell count and MTT, the ratios of G 0 /G 1 , S, G 2 /M in cell cycle were determined with flow cytometry after LDR, The cell cycle and expressions of related signal proteins were analyzed with protein assay system. Results: The results of cell count and MTT showed that there were no significant differences of proliferation rate of HCT-8 cells between 25, 50, 75, 100, 200 mGy LDR groups and sham radiation group (P>0.05); compared with high dose radiation group, there were significant differences (P 0 /G 1 phase of HCT-8 cells increased (P>0.05), the ratio of S phase decreased significantly (P 2 /M phase increased obviously (P 0 /G 1 , S, and G 2 /M phases of HCT-8 cells 48 h after radiation compared with sham radiation group (P>0.05). The protein assay result indicated that the expressions of AKt, PCNA, p27, CDK2, cyclin E, EGFR, ERK1/2, p-ERK, p-GSK-32/β in HCT-8 cells after LDR decreased compared with sham radiation group. Conclusion: LDR has no stimulating effect on HCT-8 cells. However, to some extent LDR suppress the expressions of some proteins related to proliferation and cell cycle. (authors)

  20. Absence of p53 in Clara cells favours multinucleation and loss of cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Clarke Alan R

    2002-11-01

    Full Text Available Abstract Background The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. Results p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells were proficient in S phase DNA synthesis, as determined by BrdU incorporation. However, multinucleation did not reflect altered rates of S phase synthesis, which were similar between wild type and p53-/- cultures. Nucleation defects in p53-/- Clara cells associated with increased centrosome number, as determined by confocal microscopy of pericentrin-stained cultures, and may highlight a novel role of p53 in preserving genomic integrity in lung epithelial cells. Effects of p53-deficiency were also studied following exposure to DNA damage. A p53-dependent reduction in the BrdU index was observed in Clara cells following ionizing radiation. The reduction in BrdU index in wild type cells displayed serum-dependency, and occurred only in the absence of serum. Taken together, these findings demonstrate that in murine primary Clara cell culture, cell cycle arrest is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response. Conclusion These findings highlight functions of wild type p53 protein in bipolar spindle formation, centrosome regulation, and growth control in bronchiolar Clara cells.

  1. Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

    International Nuclear Information System (INIS)

    Rutz, H. Peter; Little, John B.

    1995-01-01

    Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

  2. Effects of menstrual cycle phase on cocaine self-administration in rhesus macaques.

    Science.gov (United States)

    Cooper, Ziva D; Foltin, Richard W; Evans, Suzette M

    2013-01-01

    Epidemiological findings suggest that men and women vary in their pattern of cocaine use resulting in differences in cocaine dependence and relapse rates. Preclinical laboratory studies have demonstrated that female rodents are indeed more sensitive to cocaine's reinforcing effects than males, with estrous cycle stage as a key determinant of this effect. The current study sought to extend these findings to normally cycling female rhesus macaques, a species that shares a nearly identical menstrual cycle to humans. Dose-dependent intravenous cocaine self-administration (0.0125, 0.0250, and 0.0500 mg/kg/infusion) using a progressive-ratio schedule of reinforcement was determined across the menstrual cycle. The menstrual cycle was divided into 5 discrete phases - menses, follicular, periovulatory, luteal, and late luteal phases - verified by the onset of menses and plasma levels of estradiol and progesterone. Dependent variables including number of infusions self-administered per session, progressive ratio breakpoint, and cocaine intake were analyzed according to cocaine dose and menstrual cycle phase. Analysis of plasma hormone levels verified phase-dependent fluctuations of estradiol and progesterone, with estrogen levels peaking during the periovulatory phase, and progesterone peaking during the luteal phase. Progressive ratio breakpoint, infusions self-administered, and cocaine intake did not consistently vary based on menstrual cycle phase. These findings demonstrate that under the current experimental parameters, the reinforcing effects of cocaine did not vary across the menstrual cycle in a systematic fashion in normally cycling rhesus macaques. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun; Zheng, Lemin; Zhou, Boda; Zhang, Wei; Lv, He; Yuan, Yun

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Zheng, Lemin [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing 100191 (China); Zhou, Boda [The Department of Cardiology, Peking University Third Hospital, Beijing 100191 (China); Zhang, Wei; Lv, He [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Yuan, Yun, E-mail: yuanyun2002@sohu.com [Department of Neurology, Peking University First Hospital, Beijing 100034 (China)

    2014-03-28

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

  5. Effects of ultraviolet irradiation on the cell cycle in normal and UV-sensitive cell lines with reference to the nature of the defect in xeroderma pigmentosum variant

    International Nuclear Information System (INIS)

    Imray, P.; Mangan, T.; Saul, A.; Kidson, C.

    1983-01-01

    Analysis of the distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry has been utilized to investigate the effects of ultraviolet (UV) irradiation on cell-cycle progression in normal and UV-sensitive lymphoblastoid cell lines. In time-course studies only slight perturbation of DNA distribution was seen in normal cells, or UV-sensitive familial melanoma (FM) lines in the 48 h following irradiation. Xeroderma pigmentosum (XPA) excision-deficient cells showed a large increase in the proportion of cells in S phase 16-40 h post-irradiation. XP variant (XPV) cells were blocked in G 1 and S phases with the complete absence of cells with G 2 DNA content 16-28 h after irradiation. By 48 h post-irradiation the DNA distribution of XPA and XPV cells had returned to that of an unirradiated control. When colcemid was added to the cultures immediately after irradiation to prevent mitotic cells dividing and re-entering the cell cycle, progression through the first cycle after irradiation was followed. UV irradiation did not affect the rate of movement of cells out of G 1 into S phase in normal, FM or XPA cells. The proportion of cells in S phase was increased in UV-irradiated cultures in these cell types and the number of cells entering the G 2 +M compartment was reduced. (orig./AJ)

  6. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le

    2015-09-01

    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

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

    Science.gov (United States)

    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

  8. S phase entry of neural progenitor cells correlates with increased blood flow in the young subventricular zone.

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    Benjamin Lacar

    Full Text Available The postnatal subventricular zone (SVZ contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A(2 receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.

  9. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

    Science.gov (United States)

    Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

    2013-01-01

    We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

  10. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  11. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

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    Hyun-Ho Kwak

    2016-01-01

    Full Text Available Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC. In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin. Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

  12. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)

    Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

    2009-01-01

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  13. Dynamic interaction of p220NPAT and CBP/p300 promotes S-phase entry

    International Nuclear Information System (INIS)

    Wang, Aiyan; Ikura, Tsuyoshi; Eto, Kazuhiro; Ota, Masato S.

    2004-01-01

    Cajal bodies contain cyclin E/cdk2 and the substrate p220 NPAT to regulate the transcription of histones, which is essential for cell proliferation, however, recent mouse knockout studies indicate that cyclin E and cdk2 are dispensable for these events. Because the CBP/p300 histone acetyltransferase are also known to be involved in cell proliferation, we examined the molecular and functional interactions of p220 NPAT with the CBP/p300 at the G1/S boundary as cell cycle regulators. The subnuclear localization of p220 NPAT and CBP/p300 proteins showed that their foci partially overlapped in a cell cycle dependent manner. Overexpression of p220 NPAT and CBP/p300 cooperatively enhanced G1/S transition and DNA synthesis even without cdk2 phosphorylation site. Finally, molecular alignment analysis indicated that p220 NPAT contains several potential substrate sites for CBP/p300. Overall, our findings demonstrate that p220 NPAT and CBP/p300 form a transient complex at the G1/S boundary to play cooperative roles to promote the S-phase entry

  14. Cell cycle analysis in patients with Fanconi anemia from Serbia

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    Ćirković Sanja

    2013-01-01

    Full Text Available Fanconi anemia (FA is a rare autosomal recessive disorder, characterized by progressive bone marrow failure, chromosomal instability and cell cycle blockage in the G2 phase. The hypersensitivity of FA cells can be additionally induced with specific alkylating agents such as diepoxybutane (DEB and mitomycin C, which is used in differential diagnosis of FA. Among 72 patients with clinical suspicion of FA, who were diagnosed at the Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic” and the University Children’s Hospital (2004 - 2011, in 10 patients the diagnosis of FA was confirmed on the basis of an increased chromosome sensitivity to DEB. Five out of 10 FA patients were available for further flow cytometric analysis of cell cycle. We examined cell cycle blockage in G2 phase in untreated and with DEB treated lymphocyte cultures from FA patients and from the healthy persons, as control group. All five patients affected with FA, showed an increased DEB induced G2-phase-blockage which was over two times higher than in controls. The percentage of FA cells arrested in G2 phase was between 4,41% and 10,45% with mean value (MV of 7,76%, but in the control group this range was lower: 1,56% - 4,11% (MV: 2.84%, with no overlapping. FA patients showed an increased spontaneous arrest in G2 phase, as well, comparing to healthy controls (MV: 14,63% vs. 5,82%. Cell cycle assay of G2 phase blockage could be used as an additional diagnostic tool for confirmation of FA in patients with clinical suspicion of this disease. [Projekat Ministarstva nauke Republike Srbije, br. 173046

  15. Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

    DEFF Research Database (Denmark)

    Hagen, Lars; Kavli, Bodil; Sousa, Mirta M L

    2008-01-01

    -catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA......) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23...

  16. Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.

    Directory of Open Access Journals (Sweden)

    Ronan Broderick

    Full Text Available Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.

  17. The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase.

    Science.gov (United States)

    Cortes-Bratti, X; Chaves-Olarte, E; Lagergård, T; Thelestam, M

    1999-01-01

    The potent cytolethal distending toxin produced by Haemophilus ducreyi is a putative virulence factor in the pathogenesis of chancroid. We studied its action on eukaryotic cells, with the long-term goal of understanding the pathophysiology of the disease. Intoxication of cultured human epithelial-like cells, human keratinocytes, and hamster fibroblasts was irreversible, and appeared as a gradual distention of three- to fivefold the size of control cells. Organized actin assemblies appeared concomitantly with cell enlargement, promoted by a mechanism that probably does not involve small GTPases of the Rho protein family. Intoxicated cells did not proliferate. Similar to cells treated with other cytolethal distending toxins, these cells accumulated in the G2 phase of the cell cycle, demonstrating an increased level of the tyrosine phosphorylated (inactive) form of the cyclin-dependent kinase p34(cdc2). DNA synthesis was not affected until several hours after this increase, suggesting that the toxin acts directly on some kinase/phosphatase in the signaling network controlling the p34(cdc2) activity. We propose that this toxin has an important role both in the generation of chancroid ulcers and in their slow healing. The toxin may also be an interesting new tool for molecular studies of the eukaryotic cell- cycle machinery.

  18. Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

    Science.gov (United States)

    Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

    2018-05-01

    DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

  19. Calotropis procera extract induces apoptosis and cell cycle arrest at G2/M phase in human skin melanoma (SK-MEL-2) cells.

    Science.gov (United States)

    Joshi, Aparna L; Roham, Pratiksha H; Mhaske, Rooth; Jadhav, Mahadev; Krishnadas, Kavitha; Kharat, Amol; Hardikar, Bhagyashree; Kharat, Kiran R

    2015-01-01

    Calotropis procera (family: Asclepiadaceae) contains cardiac glycosides which are cytotoxic to cancer cells. The extracts of C. procera have been reported to be cytotoxic to many cancer cell lines and this is the first report against the human skin melanoma cells (SK-MEL-2). The SK-MEL-2 cells treated with C. procera methanolic extract (CPME) were analysed for growth inhibition and apoptosis. The exposure of phosphatidylserine in apoptotic SK-MEL-2 was analysed by using the Annexin-V FITC flow cytometry method. In CPME-treated SK-MEL-2 cells, 19.6% of apoptotic and 58.3% dead cells were observed. The 15.97% and 15.85% of early apoptotic cells were found at 20 μg/mL of the ouabain and paclitaxel, respectively. Active caspases, nuclear degradation confirmed apoptotic SK-MEL-2 cells in time- and dose-dependent manner. The cell cycle analysis shows that CPME treated cells halt at G2/M phase. Significant cytotoxic activity of CPME against SK-MEL-2 may be attributed to its high cardenolide content.

  20. Temporal remodeling of the cell cycle accompanies differentiation in the Drosophila germline.

    Science.gov (United States)

    Hinnant, Taylor D; Alvarez, Arturo A; Ables, Elizabeth T

    2017-09-01

    Development of multicellular organisms relies upon the coordinated regulation of cellular differentiation and proliferation. Growing evidence suggests that some molecular regulatory pathways associated with the cell cycle machinery also dictate cell fate; however, it remains largely unclear how the cell cycle is remodeled in concert with cell differentiation. During Drosophila oogenesis, mature oocytes are created through a series of precisely controlled division and differentiation steps, originating from a single tissue-specific stem cell. Further, germline stem cells (GSCs) and their differentiating progeny remain in a predominantly linear arrangement as oogenesis proceeds. The ability to visualize the stepwise events of differentiation within the context of a single tissue make the Drosophila ovary an exceptional model for study of cell cycle remodeling. To describe how the cell cycle is remodeled in germ cells as they differentiate in situ, we used the Drosophila Fluorescence Ubiquitin-based Cell Cycle Indicator (Fly-FUCCI) system, in which degradable versions of GFP::E2f1 and RFP::CycB fluorescently label cells in each phase of the cell cycle. We found that the lengths of the G1, S, and G2 phases of the cell cycle change dramatically over the course of differentiation, and identified the 4/8-cell cyst as a key developmental transition state in which cells prepare for specialized cell cycles. Our data suggest that the transcriptional activator E2f1, which controls the transition from G1 to S phase, is a key regulator of mitotic divisions in the early germline. Our data support the model that E2f1 is necessary for proper GSC proliferation, self-renewal, and daughter cell development. In contrast, while E2f1 degradation by the Cullin 4 (Cul4)-containing ubiquitin E3 ligase (CRL4) is essential for developmental transitions in the early germline, our data do not support a role for E2f1 degradation as a mechanism to limit GSC proliferation or self-renewal. Taken

  1. Non-homologous end joining dependency of {gamma}-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, Erich [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)]. E-mail: erich.heidenreich@meduniwien.ac.at; Eisler, Herfried [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)

    2004-11-22

    There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by {gamma}-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the {gamma}-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.

  2. Non-homologous end joining dependency of γ-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

    International Nuclear Information System (INIS)

    Heidenreich, Erich; Eisler, Herfried

    2004-01-01

    There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by γ-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the γ-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development

  3. The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca2+-dependent pathway(s)

    International Nuclear Information System (INIS)

    Manzati, Elisa; Aguiari, Gianluca; Banzi, Manuela; Manzati, Michele; Selvatici, Rita; Falzarano, Sofia; Maestri, Iva; Pinton, Paolo; Rizzuto, Rosario; Senno, Laura del

    2005-01-01

    Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca 2+ levels and activation of PKCα, thereby upregulating D1 and D3 cyclin, downregulating p21 waf1 and p27 kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca 2+ increase and PKCα activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKCα or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca 2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation

  4. Effects of [3H]UdR on the cell-cycle progression of L1210 cells

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.; Carter, S.; Kimmel, M.

    1984-01-01

    Tritium-labelled uridine (( 3 H)UdR)perturbs progression of L1210 cells through the mitotic cycle. A slowdown of G 2 cells is observed 2 hr after addition of 0.5-5.0 μci/ml of ( 3 H)UdR into cultures. At 2.5-5.0 μCi/ml of ( 3 H)UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of ( 3 H)UdR for 8-24 hr. A pulse of ( 3 H)UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G 2 slowdown) are observed when the amount of the incorporated ( 3 H)UdR is such that, on average there are fewer than thirty-six ( 3 H) decays per cell which corresponds to approximately 12-19 rads. The S-phase slowdown is seen at a dose of incorporated ( 3 H)UdR twice as high as that inducing G 2 effects. The specific localization of ( 3 H)UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed. Mathematical modelling of the cell-cycle effects of ( 3 H)UdR is provided. (author)

  5. Positive Feedback Keeps Duration of Mitosis Temporally Insulated from Upstream Cell-Cycle Events.

    Science.gov (United States)

    Araujo, Ana Rita; Gelens, Lendert; Sheriff, Rahuman S M; Santos, Silvia D M

    2016-10-20

    Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no correlation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling, we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish, variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short, constant, and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

    Science.gov (United States)

    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

  7. Transvaginal sonographic evaluation at different menstrual cycle phases in diagnosis of uterine lesions

    Directory of Open Access Journals (Sweden)

    Hajishaiha M

    2011-10-01

    Full Text Available Masomeh Hajishaiha1, Mohammad Ghasemi-rad2, Nazila Karimpour1, Nikol Mladkova3, Farzaneh Boromand11Department of Gynecology, 2Student Research Committee (SRC, Urmia University of Medical Sciences, Urmia, Islamic Republic of Iran; 3Institute of Cell and Molecular Science, London, UKPurpose: Intrauterine lesions (IULs are a common finding in women of reproductive age, particularly infertile women. Transvaginal sonography (TVS is a popular tool for IUL detection, but there are conflicting data with respect to its accuracy.Methods: Five hundred and six women were enrolled into the study. Of these, 496 underwent hysterosalpingography and subsequent TVS six different times during the course of their menstrual cycle. If a lesion was detected, it was further evaluated by sonohysterography (SHG and hysteroscopy.Results: Of 496 women, 41 were shown to have IULs by TVS and those lesions were confirmed in 39 by SHG and hysteroscopy. All 39 lesions were detectable during the ovulatory and early luteal phase (days 16–19 of the menstrual cycle. Accuracy of TVS during different phases was largely dependent on the size of the lesion. TVS falsely detected two lesions and missed fine adhesions in two patients.Conclusion: Accuracy of TVS in detection of IULs is highly dependent on the menstrual cycle phase, with the ovulatory and early luteal phase being the optimal time for this examination.Keywords: menstrual cycle phase, space occupying lesions, transvaginal sonography

  8. Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: An indication of cell cycle defects

    International Nuclear Information System (INIS)

    Hannan, Mohammed A.; Kunhi, Mohammed; Einspenner, Michael; Khan, Bashir A.; Al-Sedairy, Sultan

    1994-01-01

    In the present study, both post-irradiation DNA synthesis and G 2 phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by 3 H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G 2 phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G 2 phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G 2 accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cycle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G 2 phase accumulation developed elsewhere in characterizing AT heterozygote-like cell cycle anomaly in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle

  9. In Situ Encapsulating α-MnS into N,S-Codoped Nanotube-Like Carbon as Advanced Anode Material: α → β Phase Transition Promoted Cycling Stability and Superior Li/Na-Storage Performance in Half/Full Cells.

    Science.gov (United States)

    Liu, Dai-Huo; Li, Wen-Hao; Zheng, Yan-Ping; Cui, Zheng; Yan, Xin; Liu, Dao-Sheng; Wang, Jiawei; Zhang, Yu; Lü, Hong-Yan; Bai, Feng-Yang; Guo, Jin-Zhi; Wu, Xing-Long

    2018-04-02

    Incorporation of N,S-codoped nanotube-like carbon (N,S-NTC) can endow electrode materials with superior electrochemical properties owing to the unique nanoarchitecture and improved kinetics. Herein, α-MnS nanoparticles (NPs) are in situ encapsulated into N,S-NTC, preparing an advanced anode material (α-MnS@N,S-NTC) for lithium-ion/sodium-ion batteries (LIBs/SIBs). It is for the first time revealed that electrochemical α → β phase transition of MnS NPs during the 1st cycle effectively promotes Li-storage properties, which is deduced by the studies of ex situ X-ray diffraction/high-resolution transmission electron microscopy and electrode kinetics. As a result, the optimized α-MnS@N,S-NTC electrode delivers a high Li-storage capacity (1415 mA h g -1 at 50 mA g -1 ), excellent rate capability (430 mA h g -1 at 10 A g -1 ), and long-term cycling stability (no obvious capacity decay over 5000 cycles at 1 A g -1 ) with retained morphology. In addition, the N,S-NTC-based encapsulation plays the key roles on enhancing the electrochemical properties due to its high conductivity and unique 1D nanoarchitecture with excellent protective effects to active MnS NPs. Furthermore, α-MnS@N,S-NTC also delivers high Na-storage capacity (536 mA h g -1 at 50 mA g -1 ) without the occurrence of such α → β phase transition and excellent full-cell performances as coupling with commercial LiFePO 4 and LiNi 0.6 Co 0.2 Mn 0.2 O 2 cathodes in LIBs as well as Na 3 V 2 (PO 4 ) 2 O 2 F cathode in SIBs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard

    2015-02-01

    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  11. Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

    Science.gov (United States)

    Hashimoto, Takashi; He, Zhiwei; Ma, Wei-Ya; Schmid, Patricia C; Bode, Ann M; Yang, Chung S; Dong, Zigang

    2004-05-01

    Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

  12. Effects on proliferation and cell cycle of irradiated KG-1 cells stimulated by CM-CSF

    International Nuclear Information System (INIS)

    Guo Dehuang; Dong Bo; Wen Gengyun; Luo Qingliang; Mao Bingzhi

    2000-01-01

    In order to explore the variety of cell proliferation and cell cycle after exposure to ionizing radiation, the responses of irradiated KG-1 cells of the human myeloid leukemia stimulated by GM-CSF, the most common used cytokine in clinic, were investigated. The results showed that GM-CSF enhance KG-1 cells proliferation, reduce G0/G1 block, increase S phase and G2/M phase. The stimulation effects of the GM-CSF are more effective in irradiated group than in control group

  13. Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

    Science.gov (United States)

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

    2011-01-01

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

  14. Tousled-like kinase-dependent phosphorylation of Rad9 plays a role in cell cycle progression and G2/M checkpoint exit.

    Directory of Open Access Journals (Sweden)

    Ryan Kelly

    Full Text Available Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1 complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1 as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

  15. Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

    Science.gov (United States)

    Lange, S; Viergutz, T; Simkó, M

    2004-10-01

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

  16. Origin of the effects of cell cycle age on radiation sensitivity

    International Nuclear Information System (INIS)

    Nelson, J.M.; Braby, L.A.; Metting, N.F.; Roesch, W.C.

    1987-01-01

    The observed differences in radiation sensitivity as a function of cell age could reflect either differences in cellular capacity to remove different types of damage, or differences in damage production. The authors used centrifugal elutriation methods to produce enriched populations of G/sub 1/-, S-, and G/sub 2/-phase cells from stationary phase CHO cultures. Recognizing the imperfect correlation between cell age and cross-sectional area (the criterion upon which the separation process is based) flow-cytometric methods were used to measure cellular DNA content and determine cell age distributions. Split-dose repair kinetics were then determined for cells in different phases of the cycle. The authors find no striking differences in total repair, indicating that damage is removed at generally the same rate and with essentially the same efficiency, throughout the cell cycle. This suggests that changes in the production of damage are likely responsible for the change in sensitivity

  17. Cell flux through S phase in the mouse duodenal epithelium determined by cell sorting and radioautography

    International Nuclear Information System (INIS)

    Bjerknes, M.; Cheng, H.

    1982-01-01

    An accumulation of cells in early S phase was observed in normal mouse duodenal epithelium studied with flow cytometry. To determine if this accumulation of cells was the result of a lower rate of DNA synthesis, animals were given a single injection of 3 H-thymidine and the epithelium collected one hour later. The epithelium was processed for flow cytometry. Seven sort windows were established in different portions of the DNA histogram. Cells from each window were sorted onto glass slides that were then processed for radioautography. The number of silver grains over the nuclei of each sorted population was counted. It was found that cells in early S phase had significantly fewer grains over their nuclei than did mid- or late-S phase cells. We conclude that the accumulation of cells in early S phase is due, at least in part, to a lower rate of DNA synthesis in early than in mid or late S phase

  18. Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase.

    Science.gov (United States)

    Nakayama, Yuji; Yamaguchi, Naoto

    2005-04-01

    Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation.

  19. Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase

    International Nuclear Information System (INIS)

    Nakayama, Yuji; Yamaguchi, Naoto

    2005-01-01

    Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation

  20. Identification of a novel centrosomal protein CrpF46 involved in cell cycle progression and mitosis

    International Nuclear Information System (INIS)

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-01-01

    A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ∼ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism

  1. SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

    Energy Technology Data Exchange (ETDEWEB)

    Sadetaporn, D [Rice University, Houston, TX (United States); The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Flint, D; McFadden, C; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

    2016-06-15

    Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

  2. SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

    International Nuclear Information System (INIS)

    Sadetaporn, D; Flint, D; McFadden, C; Sawakuchi, G; Asaithamby, A

    2016-01-01

    Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

  3. Problem-Based Test: Replication of Mitochondrial DNA during the Cell Cycle

    Science.gov (United States)

    Setalo, Gyorgy, Jr.

    2013-01-01

    Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids,…

  4. Modulation of Cell Cycle Profile by Chlorella vulgaris Prevents Replicative Senescence of Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Tayyebeh Saberbaghi

    2013-01-01

    Full Text Available In this study, the effects of Chlorella vulgaris (CV on replicative senescence of human diploid fibroblasts (HDFs were investigated. Hot water extract of CV was used to treat HDFs at passages 6, 15, and 30 which represent young, presenescence, and senescence ages, respectively. The level of DNA damage was determined by comet assay while apoptosis and cell cycle profile were determined using FACSCalibur flow cytometer. Our results showed direct correlation between increased levels of damaged DNA and apoptosis with senescence in untreated HDFs (P<0.05. Cell cycle profile showed increased population of untreated senescent cells that enter G0/G1 phase while the cell population in S phase decreased significantly (P<0.05. Treatment with CV however caused a significant reduction in the level of damaged DNA and apoptosis in all age groups of HDFs (P<0.05. Cell cycle analysis showed that treatment with CV increased significantly the percentage of senescent HDFs in S phase and G2/M phases but decreased the population of cells in G0/G1 phase (P<0.05. In conclusion, hot water extract of Chlorella vulgaris effectively decreased the biomarkers of ageing, indicating its potential as an antiageing compound.

  5. Effects of (/sup 3/H)UdR on the cell-cycle progression of L1210 cells

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Z.; Carter, S.; Kimmel, M. (Memorial Sloan-Kettering Cancer Center, New York (USA))

    1984-11-01

    Tritium-labelled uridine ((/sup 3/H)UdR)perturbs progression of L1210 cells through the mitotic cycle. A slowdown of G/sub 2/ cells is observed 2 hr after addition of 0.5-5.0 ..mu..ci/ml of (/sup 3/H)UdR into cultures. At 2.5-5.0 ..mu..Ci/ml of (/sup 3/H)UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of (/sup 3/H)UdR for 8-24 hr. A pulse of (/sup 3/H)UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G/sub 2/ slowdown) are observed when the amount of the incorporated (/sup 3/H)UdR is such that, on average there are fewer than thirty-six (/sup 3/H) decays per cell which corresponds to approximately 12-19 rads. The S-phase slowdown is seen at a dose of incorporated (/sup 3/H)UdR twice as high as that inducing G/sub 2/ effects. The specific localization of (/sup 3/H)UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed. Mathematical modelling of the cell-cycle effects of (/sup 3/H)UdR is provided.

  6. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site

    DEFF Research Database (Denmark)

    Schulze, A; Zerfass, K; Spitkovsky, D

    1995-01-01

    Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation...

  7. Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

    Science.gov (United States)

    Kocsisova, Zuzana; Kornfeld, Kerry; Schedl, Tim

    2018-05-30

    The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor

  8. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    Science.gov (United States)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  9. The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

    Science.gov (United States)

    Choi, Hyun Kyung; Ryu, Hwani; Son, A-Rang; Seo, Bitna; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

    2016-04-01

    To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338. Copyright © 2016. Published by Elsevier Masson SAS.

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

    Science.gov (United States)

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

    2018-04-01

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

  11. Cell wall mannoprotein of Candida albicans induces cell cycle alternation and inhibits apoptosis of HaCaT cells via NF-κB signal pathway.

    Science.gov (United States)

    Han, Yang; Jiang, Hang-Hang; Zhang, Yu-Jing; Hao, Xing-Jia; Sun, Yu-Zhe; Qi, Rui-Qun; Chen, Hong-Duo; Gao, Xing-Hua

    2017-10-01

    Candida albicans (C. albicans) is a commensal organism in human and a well-known dimorphic opportunistic pathogenic fungus. Though plenty of researches on the pathogenesis of C. albicans have been performed, the mechanism is not fully understood. The cell wall components of C. albicans have been documented to play important roles in its pathogenic processes. To further study the infectious mechanism of C. albicans, we investigated the potential functional role of its cell wall mannoprotein in cell cycle and apoptosis of HaCaT cells. We found that mannoprotein could promote the transition of cell cycle from G1/G0 to S phase, in which Cyclin D1, CDK4 and p-Rb, the major regulators of the cell cycle progression, showed significant upregulation, and CDKN1A (cyclin dependent kinase inhibitor 1A (p21)) showed significant downregulation. Mannoprotein also could inhibit apoptosis of HaCaT cells, which was well associated with increased expression of BCL2 (Bcl-2). Moreover, mannoprotein could increase the phosphorylation levels of RELA (p65) and NFKBIA (IκBα), as the key factors of NF-κB signal pathway in HaCaT cells, suggesting the activation of NF-κB signal pathway. Additionally, a NF-κB specific inhibitor, PDTC, could rescue the effect of mannoprotein on cell cycle and apoptosis of HaCaT cells, which suggested that mannoprotein could activate NF-κB signal pathway to mediate cell cycle alternation and inhibit apoptosis. Copyright © 2017. Published by Elsevier Ltd.

  12. Establishment of human papillomavirus infection requires cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Dohun Pyeon

    2009-02-01

    Full Text Available Human papillomaviruses (HPVs are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these

  13. Analysis of the Budding Yeast Cell Cycle by Flow Cytometry.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    DNA synthesis is one of the landmark events in the cell cycle: G 1 cells have one copy of the genome, S phase cells are actively engaged in DNA synthesis, and G 2 cells have twice as much nuclear DNA as G 1 cells. Cellular DNA content can be measured by staining with a fluorescent dye followed by a flow-cytometric readout. This method provides a quantitative measurement of cell cycle position on a cell-by-cell basis at high speed. Using flow cytometry, tens of thousands of single-cell measurements can be generated in a few seconds. This protocol details staining of cells of the budding yeast Saccharomyces cerevisiae for flow cytometry using Sytox Green dye in a method that can be scaled widely-from one sample to many thousands and operating on inputs ranging from 1 million to more than 100 million cells. Flow cytometry is preferred over light microscopy or Coulter analyses for the analysis of the cell cycle as DNA content and cell cycle position are being directly measured. © 2017 Cold Spring Harbor Laboratory Press.

  14. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  15. Control points within the cell cycle

    International Nuclear Information System (INIS)

    Van't Hof, J.

    1984-01-01

    Evidence of the temporal order of chromosomal DNA replication argues favorably for the view that the cell cycle is controlled by genes acting in sequence whose time of expression is determined by mitosis and the amount of nuclear DNA (2C vs 4C) in the cell. Gl and G2 appear to be carbohydrate dependent in that cells starved of either carbohydrate of phosphate fail to make these transitions. Cells deprived of nitrate, however, fail only at Gl to S transition indicating that the controls that operate in G1 differ from those that operate in G2. 46 references, 5 figures

  16. Capsaicin induces cell cycle arrest and apoptosis in human KB cancer cells.

    Science.gov (United States)

    Lin, Chia-Han; Lu, Wei-Cheng; Wang, Che-Wei; Chan, Ya-Chi; Chen, Mu-Kuan

    2013-02-25

    Capsaicin, a pungent phytochemical in a variety of red peppers of the genus Capsicum, has shown an anti-proliferative effect on various human cancer cell lines. In contrast, capsaicin has also been considered to promote the growth of cancer cells. Thus, the effects of capsaicin on various cell types need to be explored. The anti-proliferative effects of capsaicin on human KB cancer cells are still unknown. Therefore, we examined the viability, cell cycle progression, and factors associated with apoptosis in KB cells treated with capsaicin. The cell proliferation/viability and cytotoxicity of KB cells exposed to capsaicin were determined by a sulforhodamine B colorimetric assay and trypan blue exclusion. Apoptosis was detected by Hoechst staining and confirmed by western blot analysis of poly-(ADP-ribose) polymerase cleavage. Cell cycle distribution and changes of the mitochondrial membrane potential were analyzed by flow cytometry. Furthermore, the expression of caspase 3, 8 and 9 was evaluated by immunoblotting. We found that treatment of KB cells with capsaicin significantly reduced cell proliferation/viability and induced cell death in a dose-dependent manner compared with that in the untreated control. Cell cycle analysis indicated that exposure of KB cells to capsaicin resulted in cell cycle arrest at G2/M phase. Capsaicin-induced growth inhibition of KB cells appeared to be associated with induction of apoptosis. Moreover, capsaicin induced disruption of the mitochondrial membrane potential as well as activation of caspase 9, 3 and poly-(ADP-ribose) polymerase in KB cells. Our data demonstrate that capsaicin modulates cell cycle progression and induces apoptosis in human KB cancer cells through mitochondrial membrane permeabilization and caspase activation. These observations suggest an anti-cancer activity of capsaicin.

  17. Cell Cycle Regulation by Alternative Polyadenylation of CCND1.

    Science.gov (United States)

    Wang, Qiong; He, Guopei; Hou, Mengmeng; Chen, Liutao; Chen, Shangwu; Xu, Anlong; Fu, Yonggui

    2018-05-01

    Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.

  18. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  19. Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

    International Nuclear Information System (INIS)

    Blair, O.C.; Sartorelli, A.C.

    1984-01-01

    Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35 S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35 S-sulfate and 3 H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M

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

    International Nuclear Information System (INIS)

    Su, Miaoxian; Chung, Hau Yin; Li, Yaolan

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Miaoxian [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Chung, Hau Yin, E-mail: anthonychung@cuhk.edu.hk [Biology Programme (Formally Biology Dept.), School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR (China); Li, Yaolan [Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou (China); Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Guangzhou (China)

    2011-07-29

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

  2. Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

    Science.gov (United States)

    Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

    2018-05-01

    Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

  3. Radioautographic analysis of changes in different phases of cell kinetics in murine oral mucosa

    International Nuclear Information System (INIS)

    Park, Chang Suck; You, Dong Soo

    1983-01-01

    The age related changes in the life cycle of the progenitor cell population of murine oral epithelia was studied. Using radioautographic methods which have been adopted in previous cell cycle studies, the age-related changes of different phases in renewing cells of the palatal, buccal and lingual mucosae were determined. The results confirm published findings on cell cycle changes of epithelia with aging and illustrated further that mitotic phases which has hither to been considered stationary, also changes with aging. The major parts revealed by this study are as follows: 1. The basal progenitor cells in different regions of oral mucosa have different generation times. 2. The basal cell cycle time increases as a function of aging and the region most affected by aging appears to be the epithelium of the cheek. 3. The phases of the cell cycle affected by the process of aging are in increasing order of magnitude: M-, S- and G1-phase. 4. The age related change in the number of DNA synthesizing basal progenitor cells occurs at two age periods. Between 1 and 12 months of life it decreases, while from 12 to 20 months it increases.

  4. Synchronization and Arrest of the Budding Yeast Cell Cycle Using Chemical and Genetic Methods.

    Science.gov (United States)

    Rosebrock, Adam P

    2017-01-03

    The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G 1 phase and at the M/G 1 transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G 2 /M by using hydroxyurea and nocodazole, respectively. © 2017 Cold Spring Harbor Laboratory Press.

  5. Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates

    Directory of Open Access Journals (Sweden)

    P. Borelli

    2009-06-01

    Full Text Available Protein energy malnutrition (PEM is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group were submitted to PEM with a low-protein diet (4% or were fed a control diet (20% protein ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

  6. CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

    Science.gov (United States)

    Cheong, Jit Kong; Gunaratnam, Lakshman; Hsu, Stephen I-Hong

    2008-04-25

    Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

  7. Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

    Science.gov (United States)

    Yang, X Q; Yang, J; Wang, R; Zhang, S; Tan, Q W; Lv, Q; Meng, W T; Mo, X M; Li, H J

    2015-12-02

    The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

  8. Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

    Science.gov (United States)

    Stuart, David T

    2017-01-01

    Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

  9. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Vucic V.

    2006-01-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  10. Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells

    Directory of Open Access Journals (Sweden)

    Fujita Masatoshi

    2006-10-01

    Full Text Available Abstract In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1 functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2 replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.

  11. Supercritical carbon dioxide extract of Physalis peruviana induced cell cycle arrest and apoptosis in human lung cancer H661 cells.

    Science.gov (United States)

    Wu, Shu-Jing; Chang, Shun-Pang; Lin, Doung-Liang; Wang, Shyh-Shyan; Hou, Fwu-Feuu; Ng, Lean-Teik

    2009-06-01

    Physalis peruviana L. (PP) is a popular folk medicine used for treating cancer, leukemia, hepatitis, rheumatism and other diseases. In this study, our objectives were to examine the total flavonoid and phenol content of different PP extracts (aqueous: HWEPP; ethanolic: EEPP; supercritical carbon dioxide: SCEPP-0, SCEPP-4 and SCEPP-5) and their antiproliferative effects in human lung cancer H661 cells. Among all the extracts tested, results showed that SCEPP-5 possessed the highest total flavonoid (226.19 +/- 4.15 mg/g) and phenol (100.82 +/- 6.25 mg/g) contents. SCEPP-5 also demonstrated the most potent inhibitory effect on H661 cell proliferation. Using DNA ladder and flow cytometry analysis, SCEPP-5 effectively induced H661 cell apoptosis as demonstrated by the accumulation of Sub-G1 peak and fragmentation of DNA. SCEPP-5 not only induced cell cycle arrest at S phase, it also up-regulated the expression of pro-apoptotic protein (Bax) and down-regulated the inhibitor of apoptosis protein (IAP). Furthermore, the apoptotic induction in H661 cells was found to associate with an elevated p53 protein expression, cytochrome c release, caspase-3 activation and PARP cleavage. Taken together, these results conclude that SCEPP-5 induced cell cycle arrest at S phase, and its apoptotic induction could be mediated through the p53-dependent pathway and modification of Bax and XIAP proteins expression. The results have also provided important pharmacological backgrounds for the potential use of PP supercritical fluid extract as products for cancer prevention.

  12. Regulation of the cell cycle by irradiation

    International Nuclear Information System (INIS)

    Akashi, Makoto

    1995-01-01

    The molecular mechanism of cell proliferation is extremely complex; deregulation results in neoplastic transformation. In eukaryotes, proliferation of cells is finely regulated through the cell cycle. Studies have shown that the cell cycle is regulated by s series of enzymes known as cyclin-dependent kinases (CDKs). The activities of CDKs are controlled by their association with regulatory subunits, cyclins; the expression of cyclins and the activation of the different cyclin-CDK complexes are required for the cell to cycle. Thus, the cell cycle is regulated by activating and inhibiting phosphorylation of the CDK subunits and this program has internal check points at different stages of the cell cycle. When cells are exposed to external insults such as DNA damaging agents, negative regulation of the cell cycle occurs; arrest in either G1 or G2 stage is induced to prevent the cells from prematurely entering into the next stage before DNA is repaired. Recently, a potent inhibitor of CDKs, which inhibits the phosphorylation of retinoblastoma susceptibility (Rb) gene product by cyclin A-CDK2, cyclin E-CDK2, cyclin D1-CDK4, and cyclin D2-CDK4 complexes has been identified. This protein named WAF1, Sdi1, Cip1, or p21 (a protein of Mr 21,000) contains a p53-binding site in its promoter and studies have reported that the expression of WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce WAF1. This chapter will be focused on the mechanisms of the cell cycle including inhibitors of CDKs, and the induction of WAF1 by irradiation through a pathway independent of p53 will be also described. (author)

  13. Cyclin D1 overexpression, cell cycle progression and radiosensitivity in MBP cells

    International Nuclear Information System (INIS)

    Wu Lijun; Yu Zengliang

    2000-11-01

    Clones that exhibited a minimum of 7-8 fold cyclin D1 level above the parent cell lines or the vector control were obtained after transfected with the entire coding sequence of human 1.1 kb cyclin D1 cDNA. Studies showed that there was no significant difference in Radiosensitivity between over-expressing cyclin D1 and control cultures from either mouse or human origin. Using flow cytometry to access cell cycle distribution in the exponentially growth cultures of MCF10F-D1-21 and MCF10F-V-3, it was found that there was a 50 percent increase in the proportion of G2/M phase cells and 5.3 percent decrease in the proportion of G0/G1 phase cells in MCF10F-D1-21 comparing with MCF10F-V-3, though they were with the same proportion of cells in S phase

  14. DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells.

    Science.gov (United States)

    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.

  15. Verteporfin inhibits papillary thyroid cancer cells proliferation and cell cycle through ERK1/2 signaling pathway

    Science.gov (United States)

    Liao, Tian; Wei, Wen-Jun; Wen, Duo; Hu, Jia-Qian; Wang, Yu; Ma, Ben; Cao, Yi-Min; Xiang, Jun; Guan, Qing; Chen, Jia-Ying; Sun, Guo-Hua; Zhu, Yong-Xue; Li, Duan-Shu; Ji, Qing-Hai

    2018-01-01

    Verteporfin, a FDA approved second-generation photosensitizer, has been demonstrated to have anticancer activity in various tumors, but not including papillary thyroid cancer (PTC). In current pre-clinical pilot study, we investigate the effect of verteporfin on proliferation, apoptosis, cell cycle and tumor growth of PTC. Our results indicate verteporfin attenuates cell proliferation, arrests cell cycle in G2/S phase and induces apoptosis of PTC cells. Moreover, treatment of verteporfin dramatically suppresses tumor growth from PTC cells in xenograft mouse model. We further illustrate that exposure to MEK inhibitor U0126 inactivates phosphorylation of ERK1/2 and MEK in verteporfin-treated PTC cells. These data suggest verteporfin exhibits inhibitory effect on PTC cells proliferation and cell cycle partially via ERK1/2 signalling pathway, which strongly encourages the further application of verteporfin in the treatment against PTC. PMID:29721041

  16. The PPARα/p16INK4a Pathway inhibits Vascular Smooth Muscle Cell Proliferation by repressing Cell Cycle-dependent Telomerase Activation

    Science.gov (United States)

    Gizard, Florence; Nomiyama, Takashi; Zhao, Yue; Findeisen, Hannes M.; Heywood, Elizabeth B.; Jones, Karrie L.; Staels, Bart; Bruemmer, Dennis

    2009-01-01

    Peroxisome Proliferator-Activated Receptor (PPAR) α, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARα activation suppresses G1→S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16INK4a (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARα is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARα activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect which was dependent on p16. The inhibition of cell proliferation by PPARα activation was lost in VSMC following TERT overexpression or knock-down, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARα. Finally, we demonstrate that PPARα agonists suppress telomerase activation during the proliferative response following vascular injury indicating that these findings are applicable in vivo. In concert, these results demonstrate that the anti-proliferative effects of PPARα in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade. PMID:18818403

  17. Curcumin Induces Autophagy, Apoptosis, and Cell Cycle Arrest in Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yaping Zhu

    2017-01-01

    Full Text Available Objective. Curcumin is an active extract from turmeric. The aim of this study was to identify the underlying mechanism of curcumin on PCa cells and the role of autophagy in this process. Methods. The inhibitory effect of curcumin on the growth of PANC1 and BxPC3 cell lines was detected by CCK-8 assay. Cell cycle distribution and apoptosis were tested by flow cytometry. Autophagosomes were tested by cell immunofluorescence assay. The protein expression was detected by Western blot. The correlation between LC3II/Bax and cell viability was analyzed. Results. Curcumin inhibited the cell proliferation in a dose- and time-dependent manner. Curcumin could induce cell cycle arrest at G2/M phase and apoptosis of PCa cells. The autophagosomes were detected in the dosing groups. Protein expression of Bax and LC3II was upregulated, while Bcl2 was downregulated in the high dosing groups of curcumin. There was a significant negative correlation between LC3II/Bax and cell viability. Conclusions. Autophagy could be triggered by curcumin in the treatment of PCa. Apoptosis and cell cycle arrest also participated in this process. These findings imply that curcumin is a multitargeted agent for PCa cells. In addition, autophagic cell death may predominate in the high concentration groups of curcumin.

  18. Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

    Science.gov (United States)

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

    2017-02-01

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

  19. Effects of ACTH on corticosteroid and progesterone levels in female baboons depending on the phase of the menstrual cycle

    International Nuclear Information System (INIS)

    Todua, T.N.; Goncharov, N.P.; Katsiya, G.V.; Lapin, B.A.; Vorontsov, V.I.

    1986-01-01

    To study the effect of ACTH on the endocrine function of steroid producing glands depending on the level of sex hormones in the body, a comparative study of the dynamics of steroid hormones in the follicular and luteal phases of the menstrual cycle in response to a standard does of ACTH was undertaken in experiments on hamadryad baboons. Concentrations of corticosterone, 11-deoxycortisol, and progesterone were determined in duplicate samples of plasma by radioimmunoassay. It is shown that the sensitivity of the adrenals to a single injection of ACTH is independent of the phase of the menstrual cycle and the inhibitory effects of ACTH on progesterone secretion is exhibited only in the presence of an actively functioning corpus luteus of the ovary

  20. Effects of ACTH on corticosteroid and progesterone levels in female baboons depending on the phase of the menstrual cycle

    Energy Technology Data Exchange (ETDEWEB)

    Todua, T.N.; Goncharov, N.P.; Katsiya, G.V.; Lapin, B.A.; Vorontsov, V.I.

    1986-01-01

    To study the effect of ACTH on the endocrine function of steroid producing glands depending on the level of sex hormones in the body, a comparative study of the dynamics of steroid hormones in the follicular and luteal phases of the menstrual cycle in response to a standard does of ACTH was undertaken in experiments on hamadryad baboons. Concentrations of corticosterone, 11-deoxycortisol, and progesterone were determined in duplicate samples of plasma by radioimmunoassay. It is shown that the sensitivity of the adrenals to a single injection of ACTH is independent of the phase of the menstrual cycle and the inhibitory effects of ACTH on progesterone secretion is exhibited only in the presence of an actively functioning corpus luteus of the ovary.

  1. Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

    International Nuclear Information System (INIS)

    Montano, Ryan; Thompson, Ruth; Chung, Injae; Hou, Huagang; Khan, Nadeem; Eastman, Alan

    2013-01-01

    Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence. Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models. MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G 2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval. There are two reasons why delayed

  2. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component.

    Science.gov (United States)

    Zheng, Lei; Roeder, Robert G; Luo, Yan

    2003-07-25

    We have isolated and functionally characterized a multicomponent Oct-1 coactivator, OCA-S which is essential for S phase-dependent histone H2B transcription. The p38 component of OCA-S binds directly to Oct-1, exhibits potent transactivation potential, is selectively recruited to the H2B promoter in S phase, and is essential for S phase-specific H2B transcription in vivo and in vitro. Surprisingly, p38 represents a nuclear form of glyceraldehyde-3-phosphate dehydrogenase, and binding to Oct-1, as well as OCA-S function, is stimulated by NAD(+) but inhibited by NADH. OCA-S also interacts with NPAT, a cyclin E/cdk2 substrate that is broadly involved in histone gene transcription. These studies thus link the H2B transcriptional machinery to cell cycle regulators, and possibly to cellular metabolic state (redox status), and set the stage for studies of the underlying mechanisms and the basis for coordinated histone gene expression and coupling to DNA replication.

  3. Resveratrol induces cell cycle arrest and apoptosis in human eosinophils from asthmatic individuals.

    Science.gov (United States)

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

    2016-12-01

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

  4. Cell Cycle Dependent Expression of Plk1 in Synchronized Porcine Fetal Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Anger, Martin; Kues, W. A.; Klíma, Jiří; Mielenz, M.; Kubelka, Michal; Motlík, Jan; Ešner, M.; Dvořák, P.; Carnwath, J. W.; Niemann, H.

    2003-01-01

    Roč. 65, č. 3 (2003), s. 245-253 ISSN 1040-452X R&D Projects: GA MŠk LN00A065 Grant - others:FIRCA(XX) R03-TW-05530-01 Institutional research plan: CEZ:AV0Z5045916 Keywords : Plk1 * serum deprivation * cell cycle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.543, year: 2003

  5. Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

    Science.gov (United States)

    Li, Biao; Zhao, Hong; Rybak, Paulina; Dobrucki, Jurek W; Darzynkiewicz, Zbigniew; Kimmel, Marek

    2014-09-01

    Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other

  6. Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

    International Nuclear Information System (INIS)

    Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

    2003-01-01

    Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

  7. Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle

    International Nuclear Information System (INIS)

    Nguyen, Hau; Mudryj, Maria; Guadalupe, Moraima; Dandekar, Satya

    2003-01-01

    Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G 0 /G 1 phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor

  8. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

    Directory of Open Access Journals (Sweden)

    Biddle Adrian

    2010-04-01

    Full Text Available Abstract Background Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Methods Cells isolated from fresh human head and neck carcinomas (n = 11, cell lines derived from head and neck, prostate and breast human carcinomas (n = 7, and from normal human oral mucosa (n = 5, were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin. Flow cytometry for CD44 and epithelial-specific antigen (ESA expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR. The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH and siRNA. Results In both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block

  9. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

    International Nuclear Information System (INIS)

    Harper, Lisa J; Costea, Daniela Elena; Gammon, Luke; Fazil, Bilal; Biddle, Adrian; Mackenzie, Ian C

    2010-01-01

    Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA. In both cancer biopsies and carcinoma cell lines a subset of CD44 high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44 high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44 high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. These data

  10. The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

    Science.gov (United States)

    Chandler-Brown, Devon; Schmoller, Kurt M; Winetraub, Yonatan; Skotheim, Jan M

    2017-09-25

    Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

    Science.gov (United States)

    Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

    2003-11-15

    Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Science.gov (United States)

    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

  14. Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

    Science.gov (United States)

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

    2005-08-01

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

  15. Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

    Science.gov (United States)

    Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

    2014-01-01

    CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEevasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens. PMID:25144360

  16. Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels.

    Science.gov (United States)

    Yang, Ke; Guo, Yang; Stacey, William C; Harwalkar, Jyoti; Fretthold, Jonathan; Hitomi, Masahiro; Stacey, Dennis W

    2006-08-30

    The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

  17. Response of thyroid follicular cells to gamma irradiation compared to proton irradiation. I. Initial characterization of DNA damage, micronucleus formation, apoptosis, cell survival, and cell cycle phase redistribution

    Science.gov (United States)

    Green, L. M.; Murray, D. K.; Bant, A. M.; Kazarians, G.; Moyers, M. F.; Nelson, G. A.; Tran, D. T.

    2001-01-01

    .12 Gy(-1) for protons), which suggests that the higher level of survival of gamma-irradiated cells could be attributed to the persistence of nonlethally irradiated thyrocytes and/or the capacity to repair damage more effectively than cells exposed to equal physical doses of protons. The final assessment in this study was radiation-induced cell cycle phase redistribution. Gamma rays and protons produced a similar dose-dependent redistribution toward a predominantly G(2)-phase population. From our cumulative results, it seems likely that a majority of the proton-irradiated cells would not continue to divide. In conclusion, these findings suggest that there are quantitative and qualitative differences in the biological effects of proton beams and gamma rays. These differences could be due to structured energy deposition from the tracks of primary protons and the associated high-LET secondary particles produced in the targets. The results suggest that a simple dose-equivalent approach to dosimetry may be inadequate to compare the biological responses of cells to photons and protons.

  18. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

    Science.gov (United States)

    Zakian, V A; Brewer, B J; Fangman, W L

    1979-08-01

    Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA

  19. Proliferating Cell Nuclear Antigen-dependent Rapid Recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged Sites after UV Irradiation in HeLa Cells*

    Science.gov (United States)

    Ishii, Takashi; Shiomi, Yasushi; Takami, Toshihiro; Murakami, Yusuke; Ohnishi, Naho; Nishitani, Hideo

    2010-01-01

    The licensing factor Cdt1 is degraded by CRL4Cdt2 ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G1 phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G1 phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4Cdt2, before DNA damage repair is completed. PMID:20929861

  20. Effect of hyperthermia and radiation on the cell cycle progression of HeLa cells

    International Nuclear Information System (INIS)

    Kubota, Nobuo

    1982-01-01

    The effect of hyperthermia and irradiation on cytokinetics was studied using exponentially growing HeLa cells. To determine the effect of heat and/or radiation on the cell cycle progression, the changes in the DNA distribution of the cell population after time intervals after treatment were studied. The cellular DNA content of the cell population was measured by flow cytometry. The results obtained were as follows: 1. Compared with the control, the cellular DNA content distribution of HeLa cells treated with 43 0 C for 20 min and 60 min showed cell accumulation in S and G 2 M phases 8 hours after treatment. 2. Hyperthermic treatment at 45 0 C for 20 min caused cells to accumulate in S phase in the first 4 hours and G 2 M phase after 8 to 14.5 hours, whereas heat treatment at 45 0 C for 60 min caused cells to accumulate in G 2 M phase after 24 hours. 3. Irradiation of exponentially growing cells induced a block in the progress from G 2 M to G 1 phase. 4. Dose survival curves of HeLa cells with and without postirradiation thermal treatment (43 0 C, 60 min) showed significant enhancement of radiosensitivity by hyperthermia. 5. The sequential treatment, i.e. 5 Gy irradiation followed immediately by heat treatment at 43 0 C for 60 min, caused more cells to accumulate in G 2 M phase after 24 and 48 hours, as compared with 5 Gy irradiation alone. (author)

  1. Sodium arsenite alters cell cycle and MTHFR, MT1/2, and c-Myc protein levels in MCF-7 cells

    International Nuclear Information System (INIS)

    Ruiz-Ramos, Ruben; Lopez-Carrillo, Lizbeth; Albores, Arnulfo; Hernandez-Ramirez, Raul U.; Cebrian, Mariano E.

    2009-01-01

    There is limited available information on the effects of arsenic on enzymes participating in the folate cycle. Therefore, our aim was to evaluate the effects of sodium arsenite on the protein levels of methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR) and its further relationship with the expression MT1/2 and c-myc in MCF-7 cells. Arsenite treatment (0-10 μM) for 4 h decreased MTHFR levels in a concentration-dependent fashion without significant effects on DHFR. The effects on MTHFR were observed at arsenite concentrations not significantly affecting cell viability. We also observed an increase in S-phase recruitment at all concentrations probed. Lower concentrations (< 5 μM) induced cell proliferation, showing a high proportion of BrdU-stained cells, indicating a higher DNA synthesis rate. However, higher concentrations (≥ 5 μM) or longer treatment periods induced apoptosis. Arsenite also induced dose-dependent increases in MT1/2 and c-Myc protein levels. The levels of MTHFR were inversely correlated to MT1/2 and c-Myc overexpression and increased S-phase recruitment. Our findings indicate that breast epithelial cells are responsive to arsenite and suggest that exposure may pose a risk for breast cancer. The reductions in MTHFR protein levels contribute to understand the mechanisms underlying the induction of genes influencing growth regulation, such as c-myc and MT1/2. However, further research is needed to ascertain if the effects here reported following short-time and high-dose exposure are relevant for human populations chronically exposed to low arsenic concentrations.

  2. Bioenergetic coupling between membrane transport systems and biosynthetic pathways essential for cell cycle progression

    International Nuclear Information System (INIS)

    Leister, K.J.; Cutry, A.F.; Wenner, C.E.

    1986-01-01

    Recently, it has been shown that there exists a point in the cell cycle (approximately 2 h prior to S phase entry) when (Na + /K + )ATPase pump activity is no longer needed for progression through the cycle. These data suggests that pump activity is critical in the biosynthetic processes which enables the cell to proceed through the G 1 phase. A scheme is proposed which is currently being tested that (Na + /K + )ATPase pump activity serves as the driving force in the regulation of other membrane transport processes critical for cell proliferation. For example, in post-confluent quiescent C3H-10T1/2 fibroblasts, when [K + ]/sub o/ is lowered just below the K/sub m/ of the pump for K + there is a 10-fold increase in 3 H-uridine uptake into both acid soluble and insoluble cell fractions. By modulation of the pump in this manner, glucose utilization is enhanced whereas inhibition of the pump by ouabain suppresses glucose utilization. In both methods of affecting the pump, 3 H-leucine incorporation is inhibited. Electron acceptors that influence the redox state of the cell have been shown to both stimulate or inhibit cell cycle progression. Under conditions where [K + ]/sub o/ is lowered, the nucleoside uptake responses observed were modified by electron acceptors depending on the ability to oxidize NAD(P)H directly or to interact with a cytochrome-like component, (e.g. phenazine methosulfate) reversed the enhanced uridine uptake and p-phenylene diamine further enhanced the uridine uptake response. These findings suggest that a plasma membrane redox system (presumably cyt-c like) is linked to nucleoside transport which is subject to (Na + /K + )ATPase activity

  3. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    International Nuclear Information System (INIS)

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1996-01-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation mediated by UV radiation. In these experiments, we investigated the effects of exposure to 254 nm radiation on cell cycle progression in the rabbit lens epithelial cell line N/N1003A. The RNA was harvested at various times following exposure to UV (254 nm) radiation and analyzed by dot-blot and northern blot hybridizations. These results revealed that during the first 6 h following exposure of the cells to UV, there was, associated with decreasing dose, a decrease in accumulation of transcripts specific for histones H3 and H4 and an increase in the mRNA encoding protein kinase C and β- and γ-actin. Using flow cytometry, we detected an accumulation of cells in G1/S phase of the cell cycle 1 h following exposure to 254 nm radiation. The observed changes in gene expression, especially the decreased accumulation of histone transcripts reported here, may play a role in UV-induced inhibition of cell cycle progression. (Author)

  4. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  5. Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

    International Nuclear Information System (INIS)

    Rutgers, D.H.; Niessen, D.P.P.; Linden, P.M. van der

    1987-01-01

    Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1,S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore FCM was used to monitor radiation induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tsub(c)) of approximately 84 hr, a subpopulation with a Tsub(c) of approximately 21 hr occurred. (author)

  6. The cell cycle inhibitor p27Kip¹ controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist.

    Science.gov (United States)

    Menchón, Cristina; Edel, Michael J; Izpisua Belmonte, Juan Carlos

    2011-05-01

    The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27(Kip)¹ cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27(Kip)¹ in hESC lead to a G₁phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27(Kip)¹ caused an elongated/scatter cell-like phenotype involving up-regulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27(Kip)¹ protein occupies the Twist1 gene promoter and manipulation of p27(Kip)¹ by gain and loss of function is associated with Twist gene expression changes. These results define p27(Kip)¹ expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27(Kip)¹ in controlling an epithelial to mesenchymal transition (EMT) in hESC.

  7. Umbelliferone arrest cell cycle at G0/G1 phase and induces apoptosis in human oral carcinoma (KB) cells possibly via oxidative DNA damage.

    Science.gov (United States)

    Vijayalakshmi, Annamalai; Sindhu, Ganapathy

    2017-08-01

    Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, anti-oxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC 50 - 200μM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (pKB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Galiellalactone induces cell cycle arrest and apoptosis through the ATM/ATR pathway in prostate cancer cells.

    Science.gov (United States)

    García, Víctor; Lara-Chica, Maribel; Cantarero, Irene; Sterner, Olov; Calzado, Marco A; Muñoz, Eduardo

    2016-01-26

    Galiellalactone (GL) is a fungal metabolite that presents antitumor activities on prostate cancer in vitro and in vivo. In this study we show that GL induced cell cycle arrest in G2/M phase, caspase-dependent apoptosis and also affected the microtubule organization and migration ability in DU145 cells. GL did not induce double strand DNA break but activated the ATR and ATM-mediated DNA damage response (DDR) inducing CHK1, H2AX phosphorylation (fH2AX) and CDC25C downregulation. Inhibition of the ATM/ATR activation with caffeine reverted GL-induced G2/M cell cycle arrest, apoptosis and DNA damage measured by fH2AX. In contrast, UCN-01, a CHK1 inhibitor, prevented GL-induced cell cycle arrest but enhanced apoptosis in DU145 cells. Furthermore, we found that GL did not increase the levels of intracellular ROS, but the antioxidant N-acetylcysteine (NAC) completely prevented the effects of GL on fH2AX, G2/M cell cycle arrest and apoptosis. In contrast to NAC, other antioxidants such as ambroxol and EGCG did not interfere with the activity of GL on cell cycle. GL significantly suppressed DU145 xenograft growth in vivo and induced the expression of fH2AX in the tumors. These findings identify for the first time that GL activates DDR in prostate cancer.

  9. Cell cycle dependence of boron uptake in various boron compounds used for neutron capture therapy

    International Nuclear Information System (INIS)

    Yoshida, F.; Matsumura, A.; Shibata, Y.; Yamamoto, T.; Nose, T.; Okumura, M.

    2000-01-01

    In neutron capture therapy, it is important that the tumor take boron in selectively. Furthermore, it is ideal when the uptake is equal in each tumor cell. Some indirect proof of differences in boron uptake among neoplastic cell cycles has been documented. However, no investigation has yet measured boron uptake directly. Using flow cytometry, in the present study cells were sorted by G0/G1 phase and G2/M phase, and the boron concentration of each fraction was measured with inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The results were that BSH (sodiumborocaptate) and BPA (p-boronophenylalanine) had higher rates of boron uptake in the G2/M group than in the G0/G1 group. However, in BPA the difference was more prominent, which revealed a 2.2-3.3 times higher uptake of boron in the G2/M group than in the G0/G1 group. (author)

  10. Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Bronzert, D; Vindeløv, L L

    1989-01-01

    The effects of estradiol and tamoxifen (TAM) on the estrogen-dependent human breast cancer cell line MCF-7 grown in vitro and in nude mice were compared. The effect on growth was determined by cell number in vitro and by tumor growth curves in nude mice. The effects on the cell cycle kinetics were...... determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase...... in the G1 phase from 60% to 75%. In nude mice, MCF-7 only formed tumors in estradiol-supplemented mice. No differences were observed in growth and cell kinetics between 0.1 and 1.0 mg of estradiol. Daily i.p. injections of TAM resulted in tumor growth inhibition with shrinkage of tumors. The flow...

  11. A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle-Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms.

    Science.gov (United States)

    Mancebo Quintana, J M; Mancebo Quintana, S

    2012-01-01

    The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae.

  12. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  13. Epistatic participation of REV1 and REV3 in the formation of UV-induced frameshift mutations in cell cycle-arrested yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, Erich [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)]. E-mail: erich.heidenreich@meduniwien.ac.at; Eisler, Herfried [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria); Steinboeck, Ferdinand [Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)

    2006-01-29

    Mutations arising in times of cell cycle arrest may provide a selective advantage for unicellular organisms adapting to environmental changes. For multicellular organisms, however, they may pose a serious threat, in that such mutations in somatic cells contribute to carcinogenesis and ageing. The budding yeast Saccharomyces cerevisiae presents a convenient model system for studying the incidence and the mechanisms of stationary-phase mutation in a eukaryotic organism. Having studied the emergence of frameshift mutants after several days of starvation-induced cell cycle arrest, we previously reported that all (potentially error-prone) translesion synthesis (TLS) enzymes identified in S. cerevisiae did not contribute to the basal level of spontaneous stationary-phase mutations. However, we observed that an increased frequency of stationary-phase frameshift mutations, brought about by a defective nucleotide excision repair (NER) pathway or by UV irradiation, was dependent on Rev3p, the catalytic subunit of the TLS polymerase zeta (Pol {zeta}). Employing the same two conditions, we now examined the effect of deletions of the genes coding for polymerase eta (Pol {eta}) (RAD30) and Rev1p (REV1). In a NER-deficient strain background, the increased incidence of stationary-phase mutations was only moderately influenced by a lack of Pol {eta} but completely reduced to wild type level by a knockout of the REV1 gene. UV-induced stationary-phase mutations were abundant in wild type and rad30{delta} strains, but substantially reduced in a rev1{delta} as well as a rev3{delta} strain. The similarity of the rev1{delta} and the rev3{delta} phenotype and an epistatic relationship evident from experiments with a double-deficient strain suggests a participation of Rev1p and Rev3p in the same mutagenic pathway. Based on these results, we propose that the response of cell cycle-arrested cells to an excess of exo- or endogenously induced DNA damage includes a novel replication

  14. Antitumor Activity of Tenacissoside H on Esophageal Cancer through Arresting Cell Cycle and Regulating PI3K/Akt-NF-κB Transduction Cascade

    Directory of Open Access Journals (Sweden)

    Yong-sen Jia

    2015-01-01

    Full Text Available Objective. The purpose of the study was to elucidate the molecular mechanism of tenacissoside H (TDH inhibiting esophageal carcinoma infiltration and proliferation. Methods. In vitro, EC9706 cells were treated with TDH. Cells proliferation and cell cycle were assayed. PI3K and NF-κB mRNAs expression were determined by real time PCR. In vivo, model of nude mice with tumor was established. Mice were treated with TDH. Inhibition ratio of tumor volume was calculated. PCNA expression was examined. Protein expression in PI3K/Akt-NF-κB signaling pathway was determined. Results. In vitro, TDH significantly inhibited cells proliferation in a time-and-dose-dependent manner. TDH arrested the cell cycle in S phase and significantly inhibited PI3K and NF-κB mRNA expression, compared with blank controlled group (P<0.05. In vivo, TDH strongly inhibits tumor growth and volume. PCNA expression was significantly decreased after treatment of TDH. TDH downregulated proteins expression in PI3K/Akt-NF-κB transduction cascade (P<0.05. Conclusion. TDH inhibited esophageal carcinoma infiltration and proliferation both in vitro and in vivo. The anticancer activity has relation to arresting the cell cycle at the S phase, inhibited the PCNA expression of transplanted tumors in nude mice, and regulated the protein expression in the PI3K/Akt-NF-κB transduction cascade.

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

    Science.gov (United States)

    Sukawa, Yumiko; Okamoto, Takashi

    2018-03-01

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

  16. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  17. Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

    Science.gov (United States)

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

    2016-01-01

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

  18. Staphylococcus aureus Lpl Lipoproteins Delay G2/M Phase Transition in HeLa Cells.

    Science.gov (United States)

    Nguyen, Minh-Thu; Deplanche, Martine; Nega, Mulugeta; Le Loir, Yves; Peisl, Loulou; Götz, Friedrich; Berkova, Nadia

    2016-01-01

    The cell cycle is an ordered set of events, leading to cell growth and division into two daughter cells. The eukaryotic cell cycle consists of interphase (G 1 , S, and G 2 phases), followed by the mitotic phase and G 0 phase. Many bacterial pathogens secrete cyclomodulins that interfere with the host cell cycle. In Staphylococcus aureus four cyclomodulins have been described so far that all represent toxins and are secreted into the culture supernatant. Here we show that the membrane-anchored lipoprotein-like proteins (Lpl), encoded on a genomic island called νSaα, interact with the cell cycle of HeLa cells. By comparing wild type and lpl deletion mutant it turned out that the lpl cluster is causative for the G2/M phase transition delay and also contributes to increased invasion frequency. The lipoprotein Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. Interestingly, the lipid modification, which is essential for TLR2 signaling and activation of the immune system, is not necessary for cyclomodulin activity. Unlike the other staphylococcal cyclomodulins Lpl1 shows no cytotoxicity even at high concentrations. As all Lpl proteins are highly conserved there might be a common function that is accentuated by their multiplicity in a tandem gene cluster. The cell surface localized Lpls' suggests a correlation between G2/M phase transition delay and host cell invasion.

  19. The epidermal cell kinetic response to ultraviolet B irradiation combines regenerative proliferation and carcinogen associated cell cycle delay

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, W.M.; Kirkhus, B. (Oslo Univ. (Norway))

    1989-09-01

    The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. The cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation. (author).

  20. P27 in cell cycle control and cancer

    DEFF Research Database (Denmark)

    Møller, Michael Boe

    2000-01-01

    In order to survive, cells need tight control of cell cycle progression. The control mechanisms are often lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDKs). The CDK inhibitors (CKIs) are important regulators of the CDKs. As the name implies, CKIs were...

  1. Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

    Science.gov (United States)

    Han, Guocan; Zhou, Qiang

    2016-12-01

    Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

  2. Impact of gender and menstrual cycle phase on plasma cytokine concentrations.

    LENUS (Irish Health Repository)

    O'Brien, Sinead M

    2012-02-03

    OBJECTIVE: The lifetime prevalence of major depression is twice as high in females as in males. Depression is known to increase at periods where there are changes in gonadal hormones. We examined pro- and anti-inflammatory cytokine levels during the normal menstrual cycle of healthy females compared to similar time points in healthy males. METHODS: Plasma concentrations of interleukin (IL)-4, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha) and soluble IL-6 receptor (sIL-6R) were measured with enzyme-linked immunosorbent assays in healthy females during the normal ovulatory menstrual cycle and also in males at similar time points. RESULTS: The luteal phase of the menstrual cycle is associated with increased production of sIL-6R, IL-4 and TNF-alpha compared to the early follicular phase. No change was observed in IL-6, IL-8 and IL-10 concentration throughout the menstrual cycle. We found IL-4 positively correlated with oestrogen while TNF-alpha positively correlated with progesterone. Females were found to have significantly higher concentrations of TNF-alpha and sIL-6R across all phases of the menstrual cycle, compared to males across similar time points. CONCLUSION: The normal menstrual cycle is associated with increased production of sIL-6R, IL-4 and TNF-alpha in the luteal phase compared to the early follicular phase. Females have significantly higher concentrations of sIL-6R and TNF-alpha at all time points across the menstrual cycle than males.

  3. Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging.

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    Shuya Yano

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

  4. Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

    Science.gov (United States)

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

    2016-01-01

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

  5. Cytotoxicity and Proapoptotic Effects of Allium atroviolaceum Flower Extract by Modulating Cell Cycle Arrest and Caspase-Dependent and p53-Independent Pathway in Breast Cancer Cell Lines

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    Somayeh Khazaei

    2017-01-01

    Full Text Available Breast cancer is the second leading cause of cancer death among women and despite significant advances in therapy, it remains a critical health problem worldwide. Allium atroviolaceum is an herbaceous plant, with limited information about the therapeutic capability. We aimed to study the anticancer effect of flower extract and the mechanisms of action in MCF-7 and MDA-MB-231. The extract inhibits the proliferation of the cells in a time- and dose-dependent manner. The underlying mechanism involved the stimulation of S and G2/M phase arrest in MCF-7 and S phase arrest in MDA-MB-231 associated with decreased level of Cdk1, in a p53-independent pathway. Furthermore, the extract induces apoptosis in both cell lines, as indicated by the percentage of sub-G0 population, the morphological changes observed by phase contrast and fluorescent microscopy, and increase in Annexin-V-positive cells. The apoptosis induction was related to downregulation of Bcl-2 and also likely to be caspase-dependent. Moreover, the combination of the extract and tamoxifen exhibits synergistic effect, suggesting that it can complement current chemotherapy. LC-MS analysis displayed 17 major compounds in the extract which might be responsible for the observed effects. Overall, this study demonstrates the potential applications of Allium atroviolaceum extract as an anticancer drug for breast cancer treatment.

  6. Ajwa Date (Phoenix dactylifera L. Extract Inhibits Human Breast Adenocarcinoma (MCF7 Cells In Vitro by Inducing Apoptosis and Cell Cycle Arrest.

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    Fazal Khan

    Full Text Available Phoenix dactylifera L (Date palm is a native plant of the Kingdom of Saudi Arabia (KSA and other Middle Eastern countries. Ajwa date has been described in the traditional and alternative medicine to provide several health benefits including anticholesteremic, antioxidant, hepatoprotective and anticancer effects, but most remains to be scientifically validated. Herein, we evaluated the anticancer effects of the Methanolic Extract of Ajwa Date (MEAD on human breast adenocarcinoma (MCF7 cells in vitro.MCF7 cells were treated with various concentrations (5, 10, 15, 20 and 25 mg/ml of MEAD for 24, 48 and 72 h and changes in cell morphology, cell cycle, apoptosis related protein and gene expression were studied.Phase contrast microscopy showed various morphological changes such as cell shrinkage, vacuolation, blebbing and fragmentation. MTT (2-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay demonstrated statistically significant dose-dependent inhibitions of MCF7 cell proliferation from 35% to 95%. Annexin V-FITC and TUNEL assays showed positive staining for apoptosis of MCF7 cells treated with MEAD (15 mg and 25 mg for 48 h. Flow cytometric analyses of MCF7 cells with MEAD (15 mg/ml and 20 mg/ml for 24 h demonstrated cell cycle arrest at 'S' phase; increased p53, Bax protein expression; caspase 3activation and decreased the mitochondrial membrane potential (MMP. Quantitative real time PCR (qRT-PCR analysis showed up-regulation of p53, Bax, Fas, and FasL and down-regulation of Bcl-2.MEAD inhibited MCF7 cells in vitro by the inducing cell cycle arrest and apoptosis. Our results indicate the anticancer effects of Ajwa dates, which therefore may be used as an adjunct therapy with conventional chemotherapeutics to achieve a synergistic effect against breast cancer.

  7. Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1

    DEFF Research Database (Denmark)

    Petersen, B O; Wagener, C; Marinoni, F

    2000-01-01

    is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6...... in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent...

  8. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

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    Celine J. Granier

    2014-08-01

    Full Text Available PDCD2 (programmed cell death domain 2 is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs and embryonic fibroblasts (MEFs. We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse.

  9. On the role of the phases of the mitotic cycle for the manifestation of the combined effect from the action of gamma-rays and ethylmethansulphonate in Chlorella vulgaris

    International Nuclear Information System (INIS)

    Mekhandzhiev, A.D.; Petkova, S.; Chankova, S.G.

    1978-01-01

    Synchronous cultures of Chlorella vulgaris were irradiated with gamma rays at dose of 6 and 15 kR (LD 50 , respectively LD 90 ) and immediately after that were treated with ethylmethanesulphonate (EMS). The experiments were performed at different phases of the cell cycle. The mutagenic effect was assessed on the basis of 5 basic types of mutations on the 11th day size, morphological, pigmental, mosaic and sectorial types). The combined treatment of the two mutagenic factors show - as compared to the thoretically expected - a different effect in dependence with the cell cycle phase. This effect is lower at the 0 moment of the development of the cell culture, as well as at the 16th and the 18th hours after treatment with 15 kR + EMS and 6 kR + EMS respectively. This phenomenon is probably connected with the accumulation of small physiological injuries which intensify the lethal effect. The treatment of cells in the S-phase leads to an effect somewhat higher than the summary one, while a super-additive effect was established upon treating cells with 15 kR + EMS at the 4th hour. Consequently, for the manifestation of the effect of combined treatment of importance is not only the phase of the cell cycle, but the radiation dose as well. The combined treatment at higher radiation dose tends to narrow in all cases the mutation spectrum, as compared with the combination of 6 kR + EMS. The phase of the cell culture is of importance for the broading of the induced spectrum of mutations only at the combination with the lower radiation doses. The broadest spectrum of mutations is obtained at the treatment in G 1 . Determined differences have been established also in the relation between the basis types of mutations in dependence of the cell cycle, as well as the appearance of new types of mutations, which are not to be encountered upon independent treatment of the mutagenic factors. The results obtained show the importance of the metabolic activity during the treatment of

  10. Effect of normal and tumor factors on different phases of cell populations cycle.

    Science.gov (United States)

    Inda, A M; García, A L; Errecalde, A L; Badrán, A F

    1999-12-01

    In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.

  11. Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

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    Laura L Quinn

    2014-08-01

    Full Text Available CD8+ T cell responses to Epstein-Barr virus (EBV lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE and some early (E antigens are more frequently observed than responses to epitopes of late (L expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE- and early (E-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L, interference by BILF1 increases with progression through lytic cycle (IEcycle phase-specific effects that provide mechanistic

  12. Effect of 5-fluorouracil on the cell growth and cell cycle kinetics of a mouse ascites tumor growing in vivo

    International Nuclear Information System (INIS)

    Lewin, F.; Skog, S.; Tribukait, B.; Ringborg, U.; Karolinska Sjukhuset, Stockholm

    1987-01-01

    The effect of 12, 24 and 36 mg/kg body weight doses of fluoro-uracil (5-FU) on the Bp 8 ascites sarcoma growing in vivo was studied. From sequential studies of the total number of cells together with the composition of cells in the cell cycle, the cell cycle flow was calculated and correlated to the pharmacokinetics, which was determined by using 3 He-5-FU. The dose of 12 mg/kg 5-FU affected cell growth between 24 and 72 hours, while the effect of higher doses was immediate. An early block in outflow of cells from G 1 was followed by an increased outflow, indicating an early inhibition followed by an enhancement of the initiation of the DNA synthesis. This increased outflow from G 1 together with the decrease in outflow from the early S-phase, i.e. decreased DNA synthesis, resulted in an accumulation of cells in the early part of the S-phase. The prolonged effects on the cell growth and the cell cycle flow despite the very fast decline in the drug concentration both in the ascites fluid and within the cells, together with a constant level of the drug in the macromolecular fraction, suggest an interaction between 5-FU and RNA/DNA at later times rather than an inhibition of the thymidylate synthetase activity. (orig.)

  13. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

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    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  14. Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha,25-dihydroxyvitamin D3

    International Nuclear Information System (INIS)

    Studzinski, G.P.; Bhandal, A.K.; Brelvi, Z.S.

    1985-01-01

    A recently described system for monocyte-like differentiation of HL-60 cells was utilized to determine if the initiation of this pathway can be linked to a set of replicative cellular events. The standard induction system consisted of a 4-h exposure to 100 nM 1-alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] followed by determination of nonspecific esterase and phagocytic activity 24 h later. The cell cycle status was ascertained by the incorporation of [ 3 H]thymidine and autoradiography. Studies in which cell cycle block in the G1/S phase boundary region was produced by a partial inhibition of DNA synthesis with thymidine, or sodium butyrate, showed that the exposure of such semisynchronous cultures to 1,25(OH)2D3 resulted in an increased proportion of differentiated cells. Conversely, blocking the cell cycle with vinblastine (G2/M block) or theobromine (mid-G1 block) inhibited the initiation of differentiation by 1,25(OH)2D3. Experiments in which the differentiated cells were examined for the cell cycle position at the time of the exposure to 1,25(OH)2D3 by [ 3 H]thymidine labeling and autoradiography confirmed that the late G1 and early S phase cells are those which predominate in the differentiated fraction of 1,25(OH)2D3-treated HL-60 cultures. These results link pre- and early replicative cellular events to the induction of monocytic differentiation by 1,25(OH)2D3

  15. Identification of transcription factors linked to cell cycle regulation in Arabidopsis

    OpenAIRE

    Dehghan Nayeri, Fatemeh

    2014-01-01

    Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovere...

  16. Epstein-Barr virus BGLF4 kinase retards cellular S-phase progression and induces chromosomal abnormality.

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    Yu-Hsin Chang

    Full Text Available Epstein-Barr virus (EBV induces an uncoordinated S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in S. cerevisiae. Using time-lapse microscopy, we found the fate of individual HeLa cells was determined by the expression level of BGLF4. In addition to slight cell growth retardation, BGLF4 elicits abnormal chromosomal structure and micronucleus formation in 293 and NCP-TW01 cells. In Saos-2 cells, BGLF4 induced the hyperphosphorylation of co-transfected RB, while E2F1 was not released from RB-E2F1 complexes. The E2F1 regulated activities of the cyclin D1 and ZBRK1 promoters were suppressed by BGLF4 in a dose dependent manner. Detection with phosphoamino acid specific antibodies revealed that, in addition to Ser780, phosphorylation of the DNA damage-responsive Ser612 on RB was enhanced by BGLF4. Taken together, our study indicates that BGLF4 may directly or indirectly induce a DNA damage signal that eventually interferes with host DNA synthesis and delays S-phase progression.

  17. The Concerted Action of Type 2 and Type 3 Deiodinases Regulates the Cell Cycle and Survival of Basal Cell Carcinoma Cells.

    Science.gov (United States)

    Miro, Caterina; Ambrosio, Raffaele; De Stefano, Maria Angela; Di Girolamo, Daniela; Di Cicco, Emery; Cicatiello, Annunziata Gaetana; Mancino, Giuseppina; Porcelli, Tommaso; Raia, Maddalena; Del Vecchio, Luigi; Salvatore, Domenico; Dentice, Monica

    2017-04-01

    Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation, and differentiation. The intracellular hormonal environment can be tailored by the type 1 and 2 deiodinase enzymes D2 and D3, which catalyze TH activation and inactivation respectively. In many cellular systems, THs exert well-documented stimulatory or inhibitory effects on cell proliferation; however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified. We previously showed that D3 depletion or TH treatment influences the proliferation and survival of basal cell carcinoma (BCC) cells. Surprisingly, we also found that BCC cells express not only sustained levels of D3 but also robust levels of D2. The aim of the present study was to dissect the contribution of D2 to TH metabolism in the BCC context, and to identify the molecular changes associated with cell proliferation and survival induced by TH and mediated by D2 and D3. We used the CRISPR/Cas9 technology to genetically deplete D2 and D3 in BCC cells and studied the consequences of depletion on cell cycle progression and on cell death. Cell cycle progression was analyzed by fluorescence activated cell sorting analysis of synchronized cells, and the apoptosis rate by annexin V incorporation. Mechanistic investigations revealed that D2 inactivation accelerates cell cycle progression thereby enhancing the proportion of S-phase cells and cyclin D1 expression. Conversely, D3 mutagenesis drastically suppressed cell proliferation and enhanced apoptosis of BCC cells. Furthermore, the basal apoptotic rate was oppositely regulated in D2- and D3-depleted cells. Our results indicate that BCC cells constitute an example in which the TH signal is finely tuned by the concerted expression of opposite-acting deiodinases. The dual regulation of D2 and D3 expression plays a critical role in cell cycle progression and cell death by influencing cyclin D1-mediated

  18. Cytological image of the endometrium in cows in follicular and luteal phases of the ovarian cycle and in cows with follicular and luteal ovarian cysts

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    Brodzki Piotr

    2014-03-01

    Full Text Available The experiment was conducted on 30 Holstein-Friesian cows: 10 cows in the follicular phase of the cycle and in the luteal phase 10 d later, 10 cows with follicular cysts, and 10 with luteal cysts. The presence of the ovarian structures was confirmed by ultrasonography. Serum levels of progesterone and 17β-oestradiol were tested with ELISA. Samples for cytological examination were collected from the uterus of all cows using a cytological brush. Following staining, the smears were evaluated in terms of quality and percentages of endometrial cells. In the follicular phase of the oestrous cycle, cells of type A - superficial cells (64.6 ± 4.48 were proportionally the largest group of cells. Cells of type C - basal cells (19.8 ± 2.75 were also present. In the luteal phase, the highest percentage of cells was of type B - intermediate cells (76.9 ± 4.26. When follicular cysts were present on the ovaries, the cytology resembled the follicular phase of the cycle, but with many younger type C cells (33.1 ± 4.11. In the case of luteal cysts on the ovaries, the cytology was similar to that of the luteal phase of the cycle, however with a lower percentage of type B cells (58.1 ± 5.71, and a slightly higher percentage of the other types. The differences in the cytological image of the uterus when different ovarian structures are present, depend on the hormonal activity of those structures. Due to the lack of literature data, the results of the study are important as a model, and may substantially facilitate identification of phases of the oestrus cycle, or the pathologies described, as well as indicate the current status of the endometrium

  19. Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

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    Peng Xu

    2017-03-01

    Full Text Available Human parvovirus B19 (B19V infection of primary human erythroid progenitor cells (EPCs arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2 within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

  20. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells.

    Science.gov (United States)

    Zhou, Weibo; Simpson, P Jeanette; McFadden, Jill M; Townsend, Craig A; Medghalchi, Susan M; Vadlamudi, Aravinda; Pinn, Michael L; Ronnett, Gabriele V; Kuhajda, Francis P

    2003-11-01

    C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the

  1. Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification

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    Thomas Zerjatke

    2017-05-01

    Full Text Available Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations.

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

    Science.gov (United States)

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

    2015-01-02

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

  3. Cell cycles and proliferation patterns in Haematococcus pluvialis

    Science.gov (United States)

    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.

  4. Comparative study of the antitumor effect of natural monoterpenes: relationship to cell cycle analysis

    Directory of Open Access Journals (Sweden)

    Abdeslam Jaafari

    2012-06-01

    Full Text Available Monoterpenes have been identified as responsible of important therapeutic effects of plant-extracts. In this work, we try to compare the cytotoxic effect of six monoterpenes (carvacrol, thymol, carveol, carvone, eugenol and isopulegol as well as their molecular mechanisms. The in vitro antitumor activity of the tested products, evaluated against five tumor cell lines, show that the carvacrol is the most cytotoxic monoterpene. The investigation of an eventual synergistic effect of the six natural monoterpenes with two anticancer drugs revealed that there is a significant synergy between them (p<5%. On the other hand, the effect of the tested products on cell cycle progression was examined by flow cytometry after DNA staining in order to investigate the molecular mechanism of their cytotoxic activity. The results revealed that carvacrol and carveol stopped the cell cycle progression in S phase; however, thymol and isopulegol stopped it in G0/G1 phase. Regarding carvone and eugenol, no effect on cell cycle was observed.

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

    Science.gov (United States)

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

    2010-01-01

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

  6. Cell-cycle kinetics and ultraviolet light survival in UV-1, a Chinese hamster ovary cell mutant defective in post-replication recovery

    International Nuclear Information System (INIS)

    Collins, A.

    1982-01-01

    UV-I, an ultraviolet-sensitive mutant of CHO-KI, is abnormally slow to recover from the inhibition of DNA synthesis caused by u.v. irradiation. When synchronized UV-I cells are irradiated in G 1 , their movement into S phase is unaltered, but thymidine incorporation is depressed. When irradiated in S phase, again incorporation is more depressed, and S phase suffers a greater delay in UV-I than in the parent cell. UV-I and its parent have similar capacities for excision repair of u.v.-induced damage inflicted in G 1 , and so enter S phase with similar amounts of unrepaired damage. The single-cell survival was measured after irradiation at different points in the cell cycle. The mutant and parent cells have similar values of D 0 (mean lethal dose) except in mitosis, when the parent cell shows markedly greater resistance to u.v. irradiation. Dsub(q) (quasi-threshold dose) is fairly constant for the parent cell, but in UV-I it falls to a minimum in S phase. The responses of UV-I to u.v. irradiation are generally consistent with its known defect in post-replication recovery, i.e. the ability to join up the abnormally small DNA fragments synthesized on a u.v.-damaged template. (author)

  7. Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

    Science.gov (United States)

    Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

    2014-10-01

    Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.