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PubMedDate not supplied

5-Fluorouracil induces apoptotic cell death with G2 phase arrest in human breast cancer grafted in nude mice.

Treatment with 5-fluorouracil and its derivatives causes DNA double strand breaks in the S phase, and the subsequent cell death. A 5-Fluorouracil derivative (UFT), that is converted to 5-fluorouracil in vivo, was administered orally at a dose of 18 mg/kg/day to nude mice bearing human breast cancer for 28 consecutive days. The tumors on day 7, 14, 21, and 28 after treatment were examined histopathologically and by flow cytometric cell cycle analysis. UFT treatment achieved remarkable inhibition of tumor growth, and histological examination revealed significantly less mitotic figures and more apoptotic bodies throughout the treated tumors, compared to the controls. Flow cytometric study showed changes in the cell cycle outflow of treated tumor cells, involving reduced numbers of G1 phase cells and increased numbers of S and G2 phase cells, compared to the corresponding controls. The reduced growth of tumors induced by UFT is attributable to a rise in apoptotic cell death as well as a decline in producing daughter cells, i.e. mitotic activity, and 5-fluorouracil may act cytotoxically on cancer cells via apoptosis, followed by arrest at G2 phase.

Animals↗
PubMed1991

A proposed common mechanism by which phorbol esters and epidermal growth factor delay the progression from G2 phase to mitosis of HeLa cells through phospholipid metabolites.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; also called phorbol myristate acetate) and epidermal growth factor both rapidly inhibit the transition of HeLa cells from G2 phase to mitosis in a reversible fashion with characteristics of a receptor-mediated response. It was proposed that an intracellular mediator was responsible for this inhibition. In searching for a common mediator elicited by the action of both ligands, the time course of generation of phospholipid metabolites was compared with the cell cycle response of the G2 cohort monitored by time lapse analysis. The time course and the degree of mobilization of diacylglycerols (DG) effected by TPA and by epidermal growth factor and that of phosphatidic acid correlated to a great extent with the onset and the duration of the G2 inhibition caused by both agonists. Further evidence for the proposed role of DGs and/or phosphatidic acid was obtained by the observation that membrane-penetrating 1,2-DG inhibited HeLa cells in G2 phase directly. The concentration of DG required correlated with that elicited by TPA or epidermal growth factor metabolically. This cellular response to 1,2-DG was also seen in cells after down-regulation of protein kinase C using TPA. The data indicate that cellular DG and/or phosphatidic acid may contribute to a restriction of cells in G2 phase possibly by changing membrane properties.

Arachidonic Acids↗
PubMed1984

Subpopulations of slowly cycling cells in S and G2 phase in mouse epidermis.

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, The present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G1, S and G2 phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G2 phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G2 and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G2 phase. The estimated residence times in the resting states were 38 and 32 hr in S and G2 phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.

Animals↗
PubMed1998

Distribution of camptothecin-induced break points in Chinese hamster cells treated in late S and G2 phases of the cell cycle.

The distribution of camptothecin (CPT)-induced break points in late S or G2 phase of the cell cycle observed in Chinese hamster chromosomes was analysed in 400 metaphases. Contrary to expectation, they were not localized in the heterochromatic regions, suggesting that these chromatid-type aberrations arise by a mechanism which does not involve collision of the CPT-trapped 'cleavable complex' with the replication fork. Since many break points mapped more frequently to light bands (DAPI negative) than dark bands (DAPI positive) with a frequency of 73 and 15% respectively, it could be argued that the presence of the CPT-trapped 'cleavable complex' probably interferes with chromatin condensation. In fact, the euchromatic regions, which are expected to be more actively condensed in G2 phase, were more involved in chromosomal damage. These results do not completely confirm the idea that some residual DNA synthesis occurring in G2 is responsible for the G2 clastogenic effects of CPT as the heterochromatic regions should, in this case, be more involved.

Animals↗
PubMed2003

Evaluation of the mutagenic activity of hydroxyurea on the G1-S-G2 phases of the cell cycle: an in vitro study.

Hydroxyurea is considered an antineoplastic drug, which also plays an important role in the treatment of sickle cell anemia patients. We evaluated and compared the clastogenic and cytotoxic effects of hydroxyurea, using chromosomal aberrations and mitotic index, respectively, as endpoints. In vitro short-term cultures of lymphocytes were exposed to several concentrations of this drug, at various cell cycle phases. There was a significant increase in the cytotoxicity of hydroxyurea at G1 and G1/S as well in the G2 phase of the cell cycle. Hydroxyurea did not significantly increase chromosome aberrations. There was an S-dependent cytotoxic effect of hydroxyurea, which is expected based on the known activity of hydroxyurea as an inhibitor of ribonucleotide reductase.

Analysis of Variance↗
PubMed2006

Dynamic change and derivation process of FC and DFC through G1, S and G2 phases in HeLa cells.

In order to get a deeper understanding of the relationship between nucleolus structure and its function, the dynamic change and derivation of FC (fibrillar center) and DFC (dense fibrillar component) through interphase were investigated in HeLa cells synchronized at the ultrastructural level. The results showed that there was a process of FC and DFC derivation in the nucleolus of HeLa cells during interphase. In G1 phase there were a few big FCs in the nucleolus of the HeLa cell. In S phase DFC around the FC got thickened and the configuration of the DFC changed. A lot of tiny FCs were derived from parts of the thickened DFC. We called the FC and DFC formed in G1 phase as primary FC (pri-FC) and primary DFC (pri-DFC) and the FC and DFC derived from the thickened pri-DFC as secondary FC (sec-FC) and secondary DFC (sec-DFC). In G2 phase sec-FC and sec-DFC were gradually separated from pri-DFC and scattered evenly in the nucleolus. Few large pri-FCs coexisted with numerous tiny sec-FCs in the nucleolus of HeLa cells in G2 phase. Based on the results of our observation, we suggest here a model of the dynamic change and the process of derivation of FC and DFC through interphase.

Cell Cycle↗
PubMed1990

The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product.

The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.

Amino Acid Sequence↗
PubMed1996

Exit from G2 phase after 2 Gy gamma irradiation is faster in radiosensitive human cells with high expression of the RAF1 proto-oncogene.

We have previously noted that high endogenous expression of the protein product of the full-length RAF1 proto-oncogene is related to relative intrinsic cellular radiosensitivity in 19 human cells lines in vitro. This appeared to be unrelated to the parameters of cell kinetics. In rodent and human cell lines transfected with dominant oncogenes, including Myc and MYC, Hras and HRAS and SV40, increased radioresistance has been accompanied by increased delay in progress through the G2 phase of the cell cycle after irradiation. We have thus examined the putative relationship between RAF1 expression and postirradiation perturbation of G2 phase in six of the human cell lines for which data have been reported previously. These lines exhibit a wide range of both radiosensitivity and Raf1 protein levels as measured previously by Western blotting. We report here that the cell lines whose cells appear to exit more rapidly from G2 phase are more radiosensitive (r = 0.91, P = 0.01) and express high levels of Raf1 protein (r = -0.93, P = 0.006).

Cell Line↗
PubMed2003

The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase.

In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2(+/+) and MSH2(-/-) mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2(-/-) cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

Animals↗
PubMed1993

Cytogenetic response to G2-phase X irradiation in relation to DNA repair and radiosensitivity in a cancer-prone family with Li-Fraumeni syndrome.

Noncancerous skin fibroblasts from six family members with Li-Fraumeni syndrome, five with cancer of diverse tissue origin and one with a premalignant neoplasm, showed a high frequency of chromatid aberrations, 94 to 119 breaks and 58 to 95 gaps per 100 metaphase cells arrested with colcemid 0.5 to 1.5 h after X irradiation (1.75 x 10(-2) C/kg). This response results from deficient repair of the radiation-induced DNA damage. In contrast, skin fibroblasts from two unrelated normal controls and a spouse showed 19 breaks and 17 to 19 gaps per 100 cells. Whereas all six members of the cancer-prone family had a radioresistant phenotype, only four had an inherited p53 mutation. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly (0 to 0.5 h) after G2-phase X irradiation as those from the radiosensitive control spouse. We conclude, therefore, that radiosensitivity, as determined by cell killing in asynchronous populations of skin fibroblasts, is unrelated to chromosomal sensitivity to G2-phase X irradiation. However, the persistence of a high frequency of chromatid breaks and gaps at 0.5 to 1.5 h after G2-phase X irradiation, a manifestation of deficient DNA repair, is associated with proneness to cancer in this family.

Cells, Cultured↗
PubMed1982

Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative.

A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. On the assumption that each chromatid contains a single continuous DNA double helix, chromatid breaks would represent unrepaired DNA double-strand breaks; the gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H2O2, or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

4-Nitroquinoline-1-oxide↗
PubMed2000

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.

Amsacrine↗
PubMed2001

S and G2 phase roles for Cdk2 revealed by inducible expression of a dominant-negative mutant in human cells.

Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies in Xenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G1/S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G2/M DNA contents. Several observations suggested that the latter cells were in G2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G2 arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G2 phases of the mammalian cell cycle.

Base Sequence↗
PubMed1995

Induction of senescent cell-derived inhibitor of DNA synthesis gene, SDI1, in hepatoblastoma (HepG2) cells arrested in the G2-phase of the cell cycle by 9-nitrocamptothecin.

BACKGROUND: Recent studies have demonstrated that the plant-derived alkaloid camptothecin (CPT) and its derivative, 9-nitro-CPT (9NC), are cytotoxic in tumorigenic cells but cytostatic in nontumorigenic cells in vitro and in vivo. Also, CPT induces differentiation of human leukemia cells in vitro along specific lineages. In this study, we have investigated the effects of 9NC on nontumorigenic HepG2 cells derived from human hepatoblastoma. A newly discovered senescent cell-derived inhibitor (SDI1) plays a critical role in the cell cycle, so we evaluated the effect of 9NC on the expression of the SDI1 gene. EXPERIMENTAL DESIGN: The effects of 9NC on HepG2 cells were evaluated by monitoring DNA synthesis, morphologic and ultrastructural changes of cells, and perturbation in the cell cycle and by assessing the levels of p53 protein and SDI1 mRNA. RESULTS: Treatment of HepG2 cells with 9NC results in a dose-dependent inhibition of cell proliferation and DNA synthesis. Flow cytometric analysis of DNA content showed that 9NC-treated HepG2 cells are arrested in the G2-phase of the cell cycle. Light and electron microscopic examination revealed that 9NC at low concentrations induces morphologic and growth features that resemble properties highly differentiated or senescent cells, i.e., increased cell size and decreased nuclear/cytoplasmic ratio, as well as enlarged numbers of lysosomes, mitochondria, and lipid in the cytoplasm. No significant alteration in the p53 protein level was noted in 9NC-treated cells. In contrast to untreated, logarithmically grown HepG2 cells, 9NC-treated cells arrested at the G2-phase of the cell cycle and contained increased levels of SDI1 mRNA. Kinetic studies revealed gradual increases in SDI1 mRNA synthesis. CONCLUSIONS: Induction of SDI1 mRNA by 9NC represents the first documentation that the SDI1 gene can be overexpressed in the G2-phase of the cell cycle and provides a valuable cell culture system to dissect the events controlling the G2 checkpoint. In addition, this finding corroborates the hypothesis that genes up-regulated in senescent cells can also be induced in tumor-derived immortalized cells.

Antineoplastic Agents↗
PubMed2000

A morphological study of the effect of chlorambucil during the S and G2 phases of the cell cycle of synchronized HEp-2 cancer cell populations using computerized morphometry.

Chlorambucil, a bisalkylating agent, used extensively in the treatment of autoimmune and neoplastic diseases, is known to affect DNA synthesis. However recent studies have revealed that it also affects the synthesis of other nuclear protein constituents, especially histones. Since histones play a major role in both the structural and functional integrity of chromatin, we have analyzed the morphological effects of this agent, using low dose conditions and synchronized populations of HEp-2 cancer cells in the S and G2 phases of the cell cycle. Analyses at the light and electron microscopy levels were undertaken using synchronous image analysis techniques. Computerized morphometry was used so as to evaluate various nuclear and cytological morphological parameters. It was found that chlorambucil affects the organization of chromatin, as well as other cellular parameters in a manner characteristic of decreased tumor aggressiveness. A finding of significance in this study was that chlorambucil exerted its influence on all these morphological parameters only when treatment was initiated at the beginning of the S phase and not during the second half of the S phase or the G2 phase.

Antineoplastic Agents, Alkylating↗
PubMed1988

Reversible arrest of proliferation of rat 3Y1 fibroblasts in both the G1 and G2 phases by trichostatin A.

Proliferation of rat 3Y1 cells was found to be specifically blocked by trichostatin A (TSA) at two distinct stages in the cell cycle. The first block occurred in the early G1 phase at least 9 h before the G1/S boundary, whereas the second occurred during the G2 phase. When TSA-arrested cells at the G2 phase were released from the inhibition, the cells with 4C DNA entered a new S phase without passage through the M phase, resulting in the formation of proliferative tetraploid cells. The removal of TSA induced a rapid transient increase in the transcription of c-fos and the cells required 15 h to enter the S phase after release. These results suggest that the cells arrested with TSA are quiescent (G0).

Animals↗
PubMed1997

Intact G2-phase checkpoint in cells of a human cell line lacking DNA-dependent protein kinase activity.

Cells respond to radiation-induced DNA damage in a cell cycle phase-specific manner as shown by (1) variation in radiosensitivity across the cell cycle and (2) checkpoints in G1 and G2 phase at which arrest of progression of cells through the phases of the cell cycle occurs. We studied these processes in cells of human glioma cell lines which lack (M059J(PK-)) or express (M059K(PK+)) DNA-dependent protein kinase (DNA-PK) activity. Cell populations enriched with cells of a specific cell cycle phase were y-irradiated and analyzed for cell survival. Although both cell lines were relatively sensitive in G1 phase and resistant in S phase, the differential sensitivity was greater in M059J(PK-) cells. In the studies on checkpoints, unsynchronized cells were irradiated and examined for evidence of cell cycle arrest. Neither cell line showed a postirradiation G1-phase arrest, presumably because of mutant p53 status. For M059J(PK-) cells, all doses tested (2.5-10 Gy) resulted in a significant increase in the proportion of G2/M-phase cells; however, for M059K(PK+) cells, a significant increase in G2/M phase was observed only after 10 Gy. These results suggest that the ability to activate the G2-phase checkpoint remains intact in cells which lack DNA-PK activity.

Cell Cycle↗
PubMed1996

Chromosomal radiosensitivity in G2-phase lymphocytes as an indicator of cancer predisposition.

Sanford et al. (Int. J. Radiat. Biol. 55, 963-981, 1989) have reported that G2-phase cells from many heritable cancer-prone conditions exhibit higher yields of X-ray-induced chromosome damage than those found in the majority of healthy controls. We have applied their protocol to lymphocytes of a group of control and cancer-prone individuals to see if we could confirm these observations. For control donors we observed higher aberration yields, different kinetics and more interexperiment variability than found by Sanford et al. These differences could not be attributed to unavoidable minor variations in procedures (e.g. serum batches, glassware washing methods), but the difference in X-ray qualities used in the two laboratories may have made a small contribution to the discrepancies. We attribute some of our experimental variability to the fact that, to varying extents in different experiments, centrifugation of cells prior to irradiation can slow down the progression of cells into metaphase and that cells can continue to repair during the harvesting procedure (centrifugation and hypotonic treatment). We have applied the assay to cases of ataxia telangiectasia (AT, homozygotes and heterozygotes), xeroderma pigmentosum (homozygotes and heterozygotes), familial adenomatous polyposis and the syndromes Li-Fraumeni, basal cell nevus, Down's and Fanconi's but have been unable to discriminate between these groups and controls except for AT homozygotes. By including a control sample in parallel with samples from cancer-prone groups we found a significant difference in mean aberration yields between controls and AT homozygotes and heterozygotes, but not for the other groups. Since technical features could explain the discrepancies between our laboratories, we have devised our own G2-phase assay which appears to be giving promising results.

Adenomatous Polyposis Coli↗