UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.     

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2'-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.     

Why do Neurons Enter the Cell Cycle?

Increasing evidence indicates that postmitotic, terminally differentiated neurons activate the cell cycle before death. The purpose of this cell cycle activation, however, remains elusive. In proliferating cells, cell cycle machinery is a major contributor to the DNA damage response, which is comprised of growth arrest. In quiescent cells such as terminally differentiated neurons, cell cycle-associated events may also be part of the DNA damage response. A link between DNA damage and repair, cell cycle regulation and cell death is becoming increasingly recognized for cycling cells but remains elusive for quiescent cells. Neurons are particularly susceptible to oxidative stress due to the high rate of oxidative metabolism in the brain and the low level of antioxidant enzymes compared to other somatic tissues. This is supported by fact that the intracellular end point of many neurotoxic stimuli is oxidative stress, which also represents a major cause of the neuropathology underlying a variety of neurodegenerative diseases. DNA is perhaps the major target of oxyradicals. Thus, oxidative stress may cause DNA damage, which is countered by a complex defense mechanism, the DNA damage response, which involves not only the elimination of DNA damage, but its coordination with other cellular processes such as cell-cycle progression, together directing to preserve genomic integrity. The function of such response is the removal of DNA damage by DNA repair pathways, or the elimination of damaged cells via apoptosis. The present review discusses the idea that the cell cycle machinery is a critical element of the DNA damage response not only in cycling, but also quiescent cells, and may bear the same function: to repair the damage or initiate apoptosis if the damage is too extensive to be repaired.     

Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G(1) and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.     

DNA双链断裂损伤反应及它的医学意义

DNA损伤应激反应是维持基因组稳定性的基石.细胞在长期进化中形成了由损伤监视、周期调控、损伤修复、凋亡诱导等在内的自稳平衡机制.一方面,借助感应、识别并启动精细而复杂的修复机制修复损伤;另一方面,通过DNA损伤应激活化的细胞周期检查点机制,延迟或阻断细胞周期进程,为损伤修复提供时间,使细胞能安全进入新一轮细胞周期;损伤无法修复时则诱导细胞凋亡.DNA双链断裂(double strand breaks,DSBs)是真核基因组后果最严重的损伤类型之一,其修复不利,同肿瘤等人类疾病的发生发展密切相关.新进展揭示:DSBs损伤反应信号分子ATM-Chk2-p53、H2AX等的组成性活化,是肿瘤形成早期所激活的细胞内可诱导的抗癌屏障,其信号网络的精确、精细调控在基因组稳定性维持中发挥重要作用.此外,HIV病毒整合进入宿主细胞基因组的过程也依赖于宿主细胞中ATM介导的DSBs损伤反应信号转导;ATM特异性的小分子抑制剂在抗HIV感染中显示重要的功能意义.文中重点讨论调控DSBs损伤应激反应信号网络的主要研究进展,及其在肿瘤发生、发展及抗HIV感染中的新医学意义.     

Initial cell cycle delay and depression of macromolecular synthesis of primary mouse embryo cells treated with methylnitrosourea in mass culture

Primary inbred Swiss mouse embryo cells treated with doses of methylnitrosourea which prevent their proliferation for up to 4 days show no change in their G1 and G2 cell cycle phases. They are released from the developing monolayer to a lesser extent than untreated cells. Their DNA synthesis is depressed soon after treatment and for up to 3 days. The results indicate that methylnitrosourea treatment delays the progression of mouse embryo cells in tissue culture through the cell cycle by prolonging the S phase without killing the cells.     

Reproductive death of the apoptosis type in irradiated cells of the BW5147 thymoma

The method of flow cytofluorometry and biochemical analysis were used to study the pattern and kinetics of the postirradiation death of proliferating BW5147 lymphoid cells. Irradiation with a dose of 10 Gy was shown to induce thymoma cell death by apoptosis. Radiation-induced synchronous transfer of part of cells from G1 to S-stage and blocking of all cells at G2/M stages of the cell cycle preceded the cell death. Decreasing of the growth factor content in a medium through its depletion or cultivation in conditions of low serum content accelerated cell death. A possible relationship between cell death and proliferation is discussed.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.     

The effect of caffeine on the duration of the mitotic phase cycle in CHO-K1 Chinese hamster cells, irradiated with x-rays

Alterations in the duration of mitotic cycle phases in X-irradiated Chinese hamster cells CHO K1 after caffeine (CF) treatment are studied. Delays in S- and G2-phases, induced by 1 and 5 Gr of X-irradiation, are partially or completely decreased by 1 mM or 5 mM CF, respectively. When CF is removed from the medium after irradiation, delays in S- and G2-phases are seen again, however long (0-12 hours) CF remains in the medium. The data obtained allow to suggest that since CF results in a radioresistant DNA synthesis, it may also postpone delays in S- and G2-phases, while cells are progressing through the cell cycle.     

Accumulation of a Protein Required for Division During the Cell Cycle of Escherichia coli

A heat-labile protein required for division accumulates during the duplication cycle of Escherichia coli. Its formation appears to commence shortly after the cell divides, and it reaches a maximal amount shortly before the next division. A plausible mechanism for timing cell division depends on building up the critical amount of this protein. Completion of deoxyribonucleic acid (DNA) replication is also necessary for division to occur, but it does not uniquely initiate division. The evidence for these conclusions comes from heat-shock experiments; heating to 45 C for 15 min delays division increasingly with the age of a cell. A heat shock given near the end of a cycle delays division for about 30 min, whereas at the beginning of the cycle it hardly affects division. The net result is synchronization of cell division. The effect of heat is increased in bacteria which have incorporated p-fluoro-phenylalanine into their proteins. When the incorporation is early and the heat shock is late in the cycle, division is delayed by about 30 min, indicating that the division protein is synthesized early even though its sensitivity is not observed until later. At any time in the cell cycle, heat shock simply delays total protein and DNA synthesis ((3)H-thymidine uptake) for approximately 14 min. DNA replication and cell division are thus discoordinated, since DNA replication is not synchronized by the treatment.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA.     

Cell cycle phase-specific death response of tobacco BY-2 cell line to cadmium treatment

The character of programmed cell death (PCD) in plants differs in connection with the context, triggering factors and differentiation state of the target cells. To study the interconnections between cell cycle progression and cell death induction, we treated synchronized tobacco BY-2 cells with cadmium ions that represent a general abiotic stressor influencing both dividing and differentiated cells in planta. Cadmium induced massive cell death after application in all stages of the cell cycle; however, both the progression and the forms of the cell death differed pronouncedly. Apoptosis-like PCD induced by cadmium application in the S and G2 was characterized by pronounced internucleosomal DNA fragmentation. In contrast, application of cadmium in M and G1 phases was not accompanied by DNA cleavage, indicating suppression of autolysis and non-programmed character of the death. We interpret these results in the context of the situation in planta, where the induction of apoptosis-like PCD in the S and G2 phase might be connected with a need to preserve genetic integrity of dividing meristematic cells, whereas suppression of PCD response in differentiated cells (situated in G1/G0 phase) might help to avoid death of the whole plant, and thus enable initiation of the recovery and adaptation processes.     

Transient G2M arrest and subsequent release of apoptotic and mitotic cells in vanadyl(4)-prepulsed human Chang liver cells

The relationship between cell cycling and apoptosis/programmed cell death has been perceived as either checkpoint arrests or mitotic aberration where common pathways between mitosis and apoptosis seem suggested. We show here evidence implicating both perceptions of cell cycle involvement. The process was initiated by hydroxyl free radicals (OH*) generated intracellularly from internalized vanadyl(4). Intranuclear sequestration of vanadyl(4) was verified by nuclear microscopy. Resultant high oxidative reactivity in the nucleus was shown by the redox indicator methylene blue, suggesting direct oxidative damage to genomic DNA. Oxidative stress was further enhanced by depletion of glutathione which is the main cellular reducing agent. Genomic degradation and fragmentation was confirmed by flow cytometric evaluation of terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH end-labelling (TUNEL) of DNA nicks, and cell cycle DNA profiling demonstrating sub-G1 (sub-2N) accumulation. With DNA degradation, there was a G2M transient with hyperdiploid right-shifting, consistent with G2 arrest. G2 arrest was subsequently 'released' with abolition of G2M and all other cell cycle phases except for a solitary sub-G1 (apoptotic) peak. The cytological profile of this 'release' phenomenon was initially marked by the appearance of clusters of mitotic and apoptotic cells. At later stages, the cell population was composed exclusively of nuclear ghosts, apoptotic cells, mitotic cells, and mitotic cells with both chromosomes and apoptotic condensations. Concurrent and conjoint expression of cell death and cell division as the exclusive process of an entire cell population refuted the notion of mutual exclusivity between life and death. Zn2+, an endonuclease inhibitor, abolished all observed cytological and DNA profile changes.     

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells

Ultraviolet A (UVA) radiation represents more than 90% of the UV spectrum reaching Earth's surface. Exposure to UV light, especially the UVA part, induces the formation of photoexcited states of cellular photosensitizers with subsequent generation of reactive oxygen species (ROS) leading to damages to membrane lipids, proteins and nucleic acids. Although UVA, unlike UVC and UVB, is poorly absorbed by DNA, it inhibits cell cycle progression, especially during S-phase. In the present study, we examined the role of the DNA damage checkpoint response in UVA-induced inhibition of DNA replication. We provide evidence that UVA delays S-phase in a dose dependent manner and that UVA-irradiated S-phase cells accumulate in G2/M. We show that upon UVA irradiation ATM-, ATR- and p38-dependent signalling pathways are activated, and that Chk1 phosphorylation is ATR/Hus1 dependent while Chk2 phosphorylation is ATM dependent. To assess for a role of these pathways in UVA-induced inhibition of DNA replication, we investigated (i) cell cycle progression of BrdU labelled S-phase cells by flow cytometry and (ii) incorporation of [methyl-(3)H]thymidine, as a marker of DNA replication, in ATM, ATR and p38 proficient and deficient cells. We demonstrate that none of these pathways is required to delay DNA replication in response to UVA, thus ruling out a role of the canonical S-phase checkpoint response in this process. On the contrary, scavenging of UVA-induced reactive oxygen species (ROS) by the antioxidant N-acetyl-l-cystein or depletion of vitamins during UVA exposure significantly restores DNA synthesis. We propose that inhibition of DNA replication is due to impaired replication fork progression, rather as a consequence of UVA-induced oxidative damage to protein than to DNA.     

Loss of cell cycle controls in apoptotic lymphoblasts of the bursa of Fabricius.

Lymphoblasts of the normal embryonic follicles of the chicken bursa of Fabricius undergo rapid apoptosis when exposed to gamma-radiation or when cell-cell contacts are disrupted by mechanical dispersion in short term culture. We have observed previously that overexpression of v-myc sensitizes preneoplastic bursal lymphoblasts to induction of cell death, whereas resistance to induced cell death is acquired during progression to neoplasia. In this study we observed extensive DNA degradation in the large majority of the lymphoblast population within the first hour after dispersion-induced apoptosis. Paradoxically these cells continued to progress into S-phase with the bulk of DNA cleavage and death occurring in S-phase cells (i.e., in cells with more than 2C and less than 4C DNA content). We confirmed the S phase status of apoptotic cells by determining that detection of nuclear cyclin A in individual cells also corresponded with detection of DNA breakage. Levels of cyclin E, cyclin E-dependent H1 histone kinase, and p53 proteins were maintained during dispersion-induced DNA cleavage. gamma-radiation failed either to inhibit cell cycle progression or to raise p53 levels in dispersed bursal lymphoblasts. In intact bursal follicles low doses of gamma-radiation induced p53 whereas higher, apoptosis-inducing doses failed to induce p53 or prevent G1 to S-phase progression. These results suggest that normal DNA damage-induced cell cycle checkpoint controls are lost or overridden when apoptosis is induced in bursal lymphoblasts.     

Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression

Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.     

XRCC3 Depletion Induces Spontaneous DNA Breaks and p53-Dependent Cell Death

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G2/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of γH2AX expression shows that S and G2/M phase cells express the highest fraction of γH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of γH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of γH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.