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PubMed1992

A radiation-induced inhibitor of chromosome condensation and nuclear envelope breakdown in HeLa cells.

An in vitro microscopic assay for mitosis-inducing activity in mitotic HeLa cells was developed and used to demonstrate that cells irradiated and arrested in G2 phase of the cell cycle contain an inhibitor of mitosis. This assay system has a number of advantages over other assays including the use of autologous components (HeLa nuclei and mitotic cell extracts) in contrast to the microinjection method with Xenopus oocytes and without the requirements for microinjection expertise and Xenopus oocytes. The radiation-inducible inhibitor was detected at the lowest radiation dose tested (2 Gy) with maximal activity achieved within 30 min after radiation. Inhibitor activity decayed with time after radiation (2 Gy) with no activity detected at 6 h even though the cells remained in G2 phase, suggesting that either synthesis or activation of additional components is necessary for recovery from G2 arrest. The inhibitor activity was not detected in irradiated cells treated with caffeine to induce premature recovery from G2 arrest.

Animals↗
PubMed2000

Protein tyrosine kinase inhibitor, genistein, enhances apoptosis and cell cycle arrest in K562 cells treated with gamma-irradiation.

Genistein, is a natural isoflavone compound with a potent activity against protein tyrosine kinases. The leukemic cell line, K562, is a bcr/abl (Philadelphia chromosome) positive cell line that is resistant to DNA-damaging agents, including gamma-irradiation. Treatment with genistein increased apoptosis and promoted G2-phase arrest in the non-apoptotic population of the gamma-irradiated K562 cells. Irradiated cells that survived 72 h after the irradiation had a normal distribution in cell cycle, whilst genistein treatment kept cells arrested in the G2-phase, decreased the S-phase fraction and suppressed DNA-synthesis. Taken together, our results show that genistein augments apoptotic cell death after gamma-irradiation in K562 cells and this result cannot be attributed to abrogation of the G2/M checkpoint.

Apoptosis↗
PubMed1997

DNA repair proficiency in breast cancer patients and their first-degree relatives.

Defective DNA repair capacity as measured by enumerating chromatid aberrations induced in G2 phase by X-irradiation may explain increased risk of breast cancer among relatives of patients. In the present study, chromatid damage was determined in peripheral blood lymphocytes (PBL) following in vitro exposure to 50R X-irradiation in G2 phase from 14 breast cancer (BrCa) patients, 19 first-degree relatives (FDR) of BrCa patients and 17 control women who had no family history of cancer for the last 3 generations. Controls, BrCa patients and their FDR had comparable frequency of gaps and breaks when cells were arrested with Colcemid (30 min) after X-irradiation. A steep decline in chromatid damage was observed in cells of controls when arrested after 30, 90 and 120 min of X-irradiation. BrCa patients and their FDR showed higher frequencies of lymphocytic chromatid damage as compared to controls. Chromatid damage (95 gaps + breaks per 100 cells) observed among controls at 90 min post X-irradiation was considered as the optimal level of efficient DNA repair. Thirty-five percent of controls, 93% of BrCa patients and 79% of FDR showed sub-optimal DNA repair. Amongst the FDR, the likelihood of having suboptimal DNA repair was 7 times higher and the risk of developing breast cancer was 2.7 times higher as compared to controls. Moreover, in the BrCa patients, there was frequent involvement of chromosomes 1 and 2, and chromosomes of B, D and E groups, while in FDR, involvement of chromosome 2 and chromosomes of B, D and E groups was more frequent.

Adolescent↗
PubMed1986

Cell cycle progression kinetics of regenerating mouse epidermal cells: an in vivo study combining DNA flow cytometry, cell sorting, and [3H]dThd autoradiography.

Cantharidin application to mouse skin induces cell injury followed by a regenerative wave of cells entering S phase in partial synchrony about 16 h after application. After pulse labeling with [3H]dThd the synchronized cohort of cells was traced through subsequent cell cycles during regeneration. This was accomplished by DNA flow cytometry of isolated basal cells combined with sorting from G1, S, and G2 phases followed by autoradiography at intervals after pulse labeling. Successive peaks of labeled cells in S phase at about 12-h intervals, followed by subsequent peaks in G2 and G1 phases were seen. This shows that the peaks of S-phase cells seen at 16 and 28 h after cantharidin application represent mother and daughter cells, respectively, the latter still cycling in partial synchrony. These 2 peaks of S-phase cells, therefore, are not keratinocyte subpopulations with different time lags between the stimulus to regeneration and the subsequent response. It is further shown that the mean cell cycle time is reduced from about 55 h in normal epidermis to 12 h during early regeneration. This is mainly due to a considerably reduced G1 phase duration, but the S and G2 phase durations are also reduced, although still within the range of circadian variations seen in normal animals. It is reasonable to assume a causal relationship between the considerably reduced G1 duration and loss of growth restriction. Cells with a slow progression rate through G2 phase (70% of all G2 cells) in normal mouse epidermis seem to maintain a slow progression rate during regeneration. Normal growth homeostasis seems to be gradually reestablished during the second day of regeneration.

Animals↗
PubMed2000

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase.

Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' - 5' DNA helicase identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modifications in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process. Oncogene (2000).

Adenosine Triphosphatases↗
PubMed1993

Increase of calcium levels at interphase in NIH 3T3 cells.

The fluorescent calcium ion indicator dye Fluo-3 and DNA-binding dye Hoechst 33342 were employed to determine, in a quantitative microspectrofluorometric study, the intracellular calcium ion concentration ([Ca2+]i) and the DNA content of individual living NIH3T3 cells. The well-separated excitation and emission properties of these dyes allowed us to establish for each cell both the phase of the cell cycle using DNA content and [Ca2+]i. We found that the transition from G1, through S, to the G2 phase is accompanied by a two-fold increase in [Ca2+]i. The [Ca2+]i was inhomologous in each phase of the interphase (G1, S and G2) although [Ca2+]i in the S and G2 phases was never lower than certain threshold values in the G1 and S phases respectively. [Ca2+]i in G0 cells was lower than that in G1 cells. These changes in [Ca2+]i suggest that [Ca2+]i may be an important regulator of cell cycle progression.

3T3 Cells↗
PubMed1998

Glycoconjugate and adhesion molecule changes during the cell cycle in a human embryonic epithelial cell line.

The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G1 than S and G2 phases; in the S and particularly in G2 phases the cytoplasm glycoconjugates are arranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor beta 1 integrin are well expressed in G1, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G2 than G1; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle.

Cell Adhesion↗
PubMed2001

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci.

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.

3T3 Cells↗
PubMed1991

In vitro enhancement of cis-platinum antitumor activity by caffeine and pentoxifylline in a human ovarian cell line.

Cell kinetic perturbations measuring DNA and nuclear protein with dual-parameter flow cytometry were studied in vitro after cis-platinum (DDP) treatment of a DDP-resistant human ovarian cell line (BG-1). Cell viability (trypan blue exclusion) and the cell's ability to replicate (replicating potential) are also reported. A dose-dependent G2 arrest and concomitant increase in cytotoxicity were observed. We also observed a marked increase in cytotoxicity of 2.5 micrograms/ml DDP with the addition of 1 mM of caffeine (CAF) or pentoxifylline (PTX). Additionally, the cells treated with DDP and CAF or DDP and PTX demonstrated a markedly diminished ability to replicate. Cell cycle perturbations, especially the G2-phase arrest, were markedly enhanced in the cells treated with DDP and CAF or DDP and PTX, when compared to that in cells treated with DDP alone. We conclude that the cytotoxicity of DDP is enhanced by CAF and PTX in vitro by inhibiting DNA repair during the S and G2 phases.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells.

Cell cycle checkpoints are surveillance mechanisms that monitor and coordinate the order and fidelity of cell cycle events. When defects in the division program of a cell are detected, checkpoints prevent the pursuant cell cycle transition through regulation of the relevant cyclin-cdk complex(es). Checkpoints that respond to DNA damage have been described for the G1, S and G2 phases of the cell cycle. The p53 tumour suppressor is a key regulator of G1/S checkpoints, and can promote cell cycle delay or apoptosis in response to DNA damage. The importance of these events to cellular physiology is highlighted by the fact that tumours, in which p53 is frequently mutated, have widespread defects in the G1/S DNA damage checkpoints and a heightened level of genomic instability. G2/M DNA damage checkpoints have been defined by yeast genetics, though the genes in this response are conserved in mammals. We show here using biochemical and physiological assays that p53 is dispensable for a DNA damage checkpoint activated in the G2 phase of the cell cycle. Moreover, upregulation of p53 through serine 20 phosphorylation, does not occur in G2. Conversely, we show that the Chk1 protein kinase is essential for the human G2 DNA damage checkpoint. Importantly, inhibition of Chk1 in p53 deficient cells greatly sensitizes them to radiation, validating the hypothesis of targeting Chk1 in rational drug design and development for anti-cancer therapies.

Blotting, Western↗
PubMed1994

Antagonizing effect of triphenyltin chloride on cytosine-1-beta-D-arabinofuranoside potentiation of chromosome aberrations induced by mitomycin C.

We previously reported that the organotin triphenyltin chloride (TPTC), which has been widely used as an anti-fouling coating for fishing nets and ship bottoms, potentiated clastogen-induced chromosome aberrations during the G2 phase of the cell cycle. In this communication, CHO cells treated with mitomycin C (MMC) were post-treated with TPTC in the presence and absence of other agents--cytosine-1-beta-D-arabinofuranoside (araC), hydroxyurea, or caffeine--having a similar effect during the G2 phase of the cell cycle. The potentiating effect of araC was completely inhibited in the presence of TPTC at the concentration at which TPTC showed its potentiating effect, suggesting that potentiating effects of TPTC and araC are antagonistic. On the other hand, combined treatment with TPTC and caffeine or hydroxyurea showed a potentiating effect almost equal to the sum of the potentiating effects of each given separately.

Animals↗
PubMedDate not supplied

Delayed cell cycle kinetics and chromosome aberrations induced by girostan in human peripheral blood lymphocytes (PBL) in vitro. Note 2. Chromosome aberrations.

Human peripheral blood lymphocytes (PBL) maintained in vitro for 72 hours were labeled with 5-Bromodeoxyuridine (BrdU) at 50 ug/ml and concomitantly treated with girostan at 1.5; 2 and 2.5 ug/ml during the last 13; 11; 9; 7; 5 and 3 hours before cells gathering to estimate the chromosome aberrations induced during different stages of cell cycle traverse as determined by incorporation banding pattern observed in metaphase spreads; similar untreated cultures but labeled with BrdU were set-up as controls in order to determine the phase of cell cycle traverse in the moment when girostan was given to the cultures. Chromosome aberrations were induced especially when treatments were performed 13 and 11 hours before fixation. Their frequency was rather low (up to 7% of observed metaphases) and the lesions were not dose-related. When cells were treated during S- and G2-phases, chromatid and chromosome gaps, breaks, deletions and fragments were observed. Chromatid rearrangements as tri- or quadriradials were seen only when girostan was given during middle S-phase indicating that DNA breaking and misrejoining are successive steps during the same replication round. When cells were exposed to BrdU and girostan during the last 3 hours of incubation, no labeled metaphase or structural aberration was seen, but spiralization defects and premature separation of despiralated chromatids frequently appeared. This could indicate that the induction of chromosome condensing factor and of proteins responsible for normal centromere activity is disturbed when girostan treatment is applied during the last part of G2-phase, in the immediate proximity of mitosis.

Bromodeoxyuridine↗
PubMed2006

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.

Animals↗
PubMed1993

Leptolstatin from Streptomyces sp. SAM1595, a new gap phase-specific inhibitor of the mammalian cell cycle. I. Screening, taxonomy, purification and biological activities.

Leptolstatin, a new inhibitor of the progression of G1 and G2 phases of the mammalian cell cycle, was discovered through a unique screening system, in which effects of microbial metabolites on the cell cycle progression of the cultured rat fibroblasts were monitored by flow cytometry. The new inhibitor was extracted from the fermentation broth of Streptomyces sp. SAM1595 with ethyl acetate, and purified by silica gel column chromatography and HPLC. Leptolstatin showed a strong cytostatic effect on rat normal fibroblasts 3Y1 with an IC50 value of 0.4 ng/ml, but its antimicrobial activity was very weak. A 24-hour treatment of the fibroblast cells with 10 ng/ml of leptolstatin caused an arrest at G1 or G2 phase, as determined by flow cytometry. When the G2-arrested cells were freed from leptolstatin, those containing 4C DNA entered a new S phase without intervening M phase, resulting in the formation of proliferative tetraploid cells.

Animals↗
PubMed2000

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes.

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.

Carcinoma, Transitional Cell↗
PubMed2002

Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus.

Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.

Animals↗
PubMed2005

Gene copy number and cell cycle arrest.

The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

Animals↗
PubMed2005

Temporal separation of replication and recombination requires the intra-S checkpoint.

In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block.

Cell Survival↗