Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Ras-dependent cell cycle commitment during G2 phase

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.     

Accumulation of CHO cells in G2 phase following exposure to WR-1065

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.     

Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL‐sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (q_mAb) in a dose‐dependent manner. The beneficial effect of valeric acid on culture longevity and q_mAb outweighed its detrimental effect on μ, resulting in 2.9‐fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.     

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

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 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.     

Genistein blocks breast cancer cells in the G(2)M phase of the cell cycle

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 microM genistein showed a dose-dependent accumulation in the G(2)M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G(2)M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G(2)M arrest was coupled with a parallel depletion of the G(0)/G(1) phase. To understand the mechanism of action underlying the block in G(2)M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G(2)-->M transition. As expected, p34(cdc-2) expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34(cdc-2) catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 microM genistein, cyclin B(1) (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G(2)M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34(cdc-2)/cyclin B(1) complex only in all of the studied cell lines.     

Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

The in vivo arrest of Ehrlich ascitic carcinoma cells in the G2 phase of the cell cycle

Evidence for arrested G2 cell subpopulation in the Ehrlich ascite carcinoma has been provided using flow cytometry stathmokinetic and bromdesoxyuridine--Hoechst 33258 techniques. After exposure in DME--F-12 media G2-arrested cells synchronically enter mitosis and then move trough G1 period. Durations have been measured for G1. G2 phases and G2-G1 transition.     

Ecdysone-induced accumulation of mosquito cells in the G1 phase of the cell cycle

We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with Drosophila melanogaster cells, treatment of C7-10 cells with 20E inhibits proliferation. In the presence of 10⁻⁶ M 20E, a gradual decline in cell number is typically apparent at 24 h. Media components such as phenol red and the potential presence of endogenous steroids in serum have no effect on the response to 20E. Pre-treating the cells with 10⁻⁸ M 20E, with or without an intervening hormone-free period, did not alter the response to 10⁻⁶ M 20E. However, replenishment of the medium appeared to synchronize the response to 10⁻⁶ M 20E, causing an abrupt and complete cessation of cell division by 48 h. Flow cytometry over a 20 h period showed a decrease in the proportion of cells in S within 4-6 h after exposure to 20E. By 6-10 h, a transient increase in G2 was followed by the accumulation of more than 70% of the cells in G1. These data suggest that after treatment with 20E, cells complete the ongoing cycle before arresting in G1. Consistent with the decrease in the proportion of cells in S and G2, western blots show that levels of cyclin A, which is required during the S phase of the cycle, decreased in 20E-treated cells.     

Hydroxyurea treatment affects the G1 phase in next generation CHO cells

DNA replication kinetics were studied in populations of synchronized CHO cells treated in the previous generation with hydroxyurea. These CHO cells were re-synchronized by selective detachment of mitotic cells after previously synchronized G1 traversing cultures were treated with 0.1 mM and 2 mM hydroxyurea for 9 and 13 h. Our results show that these cells exhibit a shortening of G1 of at least 1 h relative to cells selected in mitosis from untreated exponentially growing cultures. Survival studies indicated that the hydroxyurea treatments did not affect plating efficiencies. Cell viability was reduced when the initially synchronized populations were blocked with 2 mM, but not 0.1 mM hydroxyurea for greater than 13 h. DNA replication measurements after these blocks showed that all cultures treated with 2 mM hydroxyurea for either 9, 13 or 15 h were blocked at the same point near the G1/S boundary, and then progressed through S phase with similar kinetics. The observed shortening of G1 in the next generation of these cells was independent of both the concentration (0.1 or 2.0 mM) and the time (9 or 13 h) of the hydroxyurea block. These results suggest that specific events relating to the next cell generation can be uncoupled from DNA synthesis and can occur when hydroxyurea inhibits normal cell cycle traverse of G1 cells into and through S phase.     

Phospholipids are synthesized in the G2/M phase of the cell cycle

Very little is known about the metabolism of phospholipids in the G2 and M phases of the cell cycle, but limited studies have led to the postulation that phospholipid synthesis ceases during this period. To investigate whether phospholipids are synthesized in the G2/M phase of the cell cycle, protocols were developed to produce synchronized MCF-7 cell populations with greater than 80% of the cells in G1/S or G2/M phases that moved in synchrony following removal of the blocking agent. Analysis of the activities of key phosphatidylcholine and phosphatidylethanolamine biosynthetic enzymes in subcellular fractions obtained from MCF-7 cells at different cell cycle phases revealed that there was robust activity of key enzymes in the fractions prepared from MCF-7 cells in G2/M phase. Radiolabeled choline and ethanolamine were rapidly incorporated into cells maintained at G2/M phase with nocodazole, and the rates of incorporation were similar to those obtained in cells allowed to progress into the G1 phase. Furthermore, radiolabeled glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid in MCF-7 cells maintained at G2/M phase with nocodazole. Similar results were obtained in CHO cells. These results demonstrate that glycerophospholipid synthesis is very active in the G2/M phase of these cells. Therefore, the postulated cessation of phospholipid synthesis in G2/M phases is not applicable to all cell types.     

Cytophotometric evaluation of the transition of cells from the G0 phase to the G1 phase of the cell cycle

Feulgen stained nuclei of PHA-stimulated human blood lymphocytes were used for cytophotometric chromatin pattern analysis. Similar distributions of low optical density values indicating the predominance of diffuse chromatin were obtained for G1, S and G2 cells. Condensed chromatin was predominant in G0 and M nuclei. Integral versus average optical densities scatter plots analyses permitted one to distinguish cells undergoing different phases of cell cycle including G0 and G1.     

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.     

Adhesion and the cell cycle in cultured L929 and CHO cells

The adhesiveness of L929 and CHO-K1 cells was monitored throughout their respective cell cycles. The cell cycle stages were identified by a combination of measuring, histochemical and autoradiographic techniques. Adhesiveness was shown to be maximal during the S and late G2 phases, and minimal at mitosis. S cells adhered selectively to other S cells, while late G2 phase cells adhered selectively to other late G2 cells. Mixing of the two cell lines, L929 and CHO-K1, resulted in the S phase cells adhering to each other irrespective of the cell line to which they belonged. The phases of increased adhesiveness and selectivity coincided with a decrease in cell surface negative charge, corresponding to well documented changes in the morphology of the cells.     

Centipedegrass extract enhances radiosensitivity in melanoma cells by inducing G2/M cell cycle phase arrest

Molecular Biology Reports - Melanoma is aggressive, highly metastatic, and potentially fatal. In the case of patients with advanced melanoma, it is difficult to expect a good prognosis, since this...     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.