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PubMed2001

G2 phase cell cycle arrest in human skin following UV irradiation.

The contribution of the short wavelength ultraviolet (UV) component of sunlight to the aetiology of skin cancer has been widely acknowledged, although its direct contribution to tumour initiation or progression is still poorly understood. The loss of normal cell cycle controls, particularly checkpoint controls, are a common feature of cancer. UV radiation causes both G1 and G2 phase checkpoint arrest in vitro cultured cells. In this study we have investigated the cell cycle responses to suberythemal doses of UV on skin. We have utilized short-term whole organ skin cultures, and multi parameter immunohistochemical and biochemical analysis to demonstrate that basal and suprabasal layer melanocytes and keratinocytes undergo a G2 phase cell cycle arrest for up to 48 h following irradiation. The arrest is associated with increased p16 expression but no apparent p53 involvement. This type of organ culture provides a very useful model system, combining the ease of in vitro manipulation with the ability to perform detailed molecular analysis in a normal tissue environment.

Bromodeoxyuridine↗

PubMed1992

Enhanced levels of radiation-induced G2 phase delay in ataxia telangiectasia heterozygotes.

Ataxia telangiectasia (AT) is a multiform genetic disease characterized by immunodeficiency, cerebellar abnormalities, and cancer predisposition. Heterozygotes also have an increased risk of developing several different cancers. It has been estimated that as many as 18% of all patients with breast cancer, the cancer most clearly associated with AT heterozygotes, may be carriers of the AT gene. We describe an assay for AT heterozygotes that relies on the previous observation that cells from AT homozygotes show a greater and more prolonged radiation-induced accumulation in the G2 phase of the cell cycle than do normal controls. We showed that all 6 A-T heterozygotes show a greater extent of G2 phase delay at different times postirradiation than do controls. The degree of accumulation was less than that observed in AT homozygotes. Only two of 22 controls showed overlap with heterozygotes at 18 hours postirradiation, and that number was reduced to one at the 24-hour point. As a group, AT heterozygotes were intermediate between controls and AT homozygotes at both time points after irradiation. This assay is relatively simple and reliable and can be performed in any laboratory with access to both Epstein-Barr virus (EBV) for transformation of lymphocytes and a fluorescence-activated cell analyzer.

Ataxia Telangiectasia↗

PubMed1994

Comparative study of radiation-induced G2 phase delay and chromatid damage in families with ataxia-telangiectasia.

Two assay systems, radiation-induced chromosome aberrations and flow cytometry, were compared for the detection of ataxia-telangiectasia (A-T) heterozygotes. In three A-T families, the frequencies of chromatid aberrations in phytohemagglutinin-stimulated blood lymphocytes after 1 Gy of gamma-irradiation were twofold higher in A-T homozygotes than in obligate A-T heterozygotes, which were in turn approximately twofold higher than in normal control cells. Other consanguineous relatives of A-T patients had intermediate levels of induced chromatid aberrations, suggesting that they were carriers of the gene. Matched Epstein-Barr virus-transformed lymphoblastoid cell lines from A-T homozygotes showed a greater radiation-induced accumulation in the G2 phase of the cell cycle than did control cells. In family B, both obligate heterozygotes had increased G2 delay, as did the one heterozygote available for family C, and two of the grandparents in that family were in the high range for G2 delay. Neither parent in family A had high G2 phase delay after irradiation although the induced chromatid aberrations were in the heterozygote valve range. These results show a good concordance between the two assay systems for A-T heterozygotes, with the chromatid aberrations somewhat more consistent.

Adult↗

PubMed2000

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle.

PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.

Catalysis↗

PubMed1989

The lack of correlation between the rate of rRNA transport from nucleoli into cytoplasm and the duration of G1 and G2 phases in root meristem cells.

In root meristems of 3 species (Secale cereale L., Vicia faba L. subsp. minor, Allium cepa L.) the durations of cell cycles and their phases were calculated using 3H-thymidine labelling. In the above species and in Helianthus annuus L. (parameters of the cell cycle determined earlier) the G1 and G2 phase durations were different: G1 + 1/2 M from 3 h to 6.1 h, G2 + 1/2 M from 1.1. h to 8.3 h, depending on the species. The rate of rRNA transport from nucleoli into cytoplasm during recovery after cold treatment was calculated from our data presented earlier. The results indicate that in 4 species studied there is no correlation (at P = 0.05) between the rate of rRNA transport and the duration of G1 and G2 phases.

Cell Cycle↗

PubMed1998

A novel p53-inducible gene coding for a microtubule-localized protein with G2-phase-specific expression.

Wild-type (wt) p53 can act as a sequence-specific transcriptional activator and it is believed that p53 elicits at least part of its biological effects by regulating the expression of specific target genes. By using a differential subtractive hybridization approach in a murine cell line stably transfected with a temperature-sensitive p53 mutant (Val135), we isolated a set of genes markedly induced by wt p53. One of them, provisionally named B99, was further characterized; its transcriptional induction was dependent on wt p53 function and the corresponding protein product was shown to accumulate after DNA damage in different cell types. Immunofluorescence analysis located the B99 protein to the microtubule network. Flow cytometry revealed that upon activation of p53 function the endogenous B99 protein was selectively induced in the G2 fraction of the cell population. When B99 was ectopically expressed in p53-null murine fibroblasts, B99-transfected cells displayed an increased fraction with a 4N DNA content, indicative of interference with G2 phase progression. Taken together these data suggest that B99 might play a role in mediating specific biological activities of wt p53 during the G2 phase.

Amino Acid Sequence↗

PubMedDate not supplied

Clastogenic effects induced in vitro by CCNU and AMD during G2-phase in bone marrow cells of Wistar rats.

Three concentrations of CCNU and AMD (1, 5 and 10 micrograms/ml) were given to bone marrow cells of Wistar rats immediately after their removal from the body into the culture medium. Abnormal metaphases observed 2, 3 and 4 h later were considered to represent the effect induced by the two drugs on G2-phase chromosomes in a cell population that traversed in vivo previous phases of the cell cycle. In order to determine if the cells examined at metaphase completed S-phase in vivo, BroU at 100 micrograms/ml was added into the culture medium and metaphases displaying replication banding were scored 2, 3 and 4 hours afterwards. Only 4% of metaphases with incorporated BrdU (Bromodeoxyuridine) were seen after 4 hours exposure and none after 2 or 3 hours, indicating that treatment was given to cells during G2-phase. A dose-related clastogenic action was observed both after AMD and CCNU treatments, but the response was essentially determined by the lapse of time between treatment and mitosis. The highest chromosome damaging effect was induced by CCNU when administered 4 hours before mitosis, while AMD reached the highest clastogenic effectiveness when given 2 hours before mitosis. These results express G2 sensitivity of bone marrow cells that traversed G1- and S-phases in vivo and consequently possessed a biochemical "machinery" initiated and effective into the rat body. The capability of primary cultures of bone marrow cells to mimic the mechanisms of the in vivo clastogenic action is discussed.

Animals↗

PubMed1997

Detection of heterozygous carriers of the ataxia-telangiectasia (ATM) gene by G2 phase chromosomal radiosensitivity of peripheral blood lymphocytes.

In ataxia-telangiectasia (A-T) patients, mutations in a single gene, ATM, result in an autosomal recessive syndrome that embraces a variety of clinical features and manifests extreme radiosensitivity and a strong pre-disposition to malignancy. Heterozygotes for the ATM gene have no clinical expression of A-T but may be cancer prone with a moderate increase in in vitro radiosensitivity. We performed a blind chromosomal analysis on G2-phase lymphocytes from 7 unrelated A-T patients, 13 obligate A-T heterozygotes (parents of the patients), and 14 normal controls following X-irradiation with 1 Gy in order to evaluate this cytogenetic method as a tool for detection of ATM carriers. Both A-T homozygotes and heterozygotes showed significantly increased levels of radiation-induced chromatid damage relative to that of normal controls. These results show that the G2-phase chromosomal radiosensitivity assay can be used for the detection of A-T heterozygotes. In combination with molecular genetic analyses, this test may be of value in studies of familial and sporadic cancers aimed at determination of the potential involvement of ATM mutations in tumor risk or development.

Adolescent↗

PubMed1996

The presence of DNA breaks and the formation of chromatid aberrations after incorporation of 125IdUrd may be necessary but are not sufficient to block cell cycle progression in G2 phase.

Cell progression into mitosis and chromatid aberration frequencies were compared in two Chinese hamster ovary (CHO) cell lines after incorporation of 125IdUrd. Asynchronous, exponentially growing populations of CHO K1 and the DNA repair-deficient, radiation-sensitive CHO irs-20 cells were compared after a 10-min exposure to 14.8 kBq/ml 125IdUrd. Essentially no differences were seen for either end point between the cells of the two cell lines. As the cells in S phase at the time of labeling entered the mitotic cell selection window, the number of mitotic cells of each cell line declined to approximately 60% of the respective unlabeled control. Chromosome analysis of the mitotically selected cells indicated an 125I decay-dependent increase in the number of chromatid aberrations in cells of both cell lines. The appearance of aberrations together with the known rates of production and rejoining of DNA double-strand breaks show that cells are able to progress through G2 phase and into mitosis in the presence of such breaks. The data suggest that DNA damage may be necessary, but is not sufficient to cause a radiation-induced blockade of cell progression through G2 phase.

Animals↗

PubMed2003

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

CDC2 Protein Kinase↗

PubMed1990

Epidermal growth factor inhibits transiently the progression from G2-phase to mitosis: a receptor-mediated phenomenon in various cells.

An immediate effect of epidermal growth factor (EGF) on the cell cycle is described. EGF, when given to replicating cells such as HeLa, A431, and D HER 14, very rapidly inhibits the transition from G2 phase to mitosis (M) in a transient fashion. The influence of EGF (10(-10) to 10(-7) M concentrations) on the G2-M transition of individual cells has been analyzed by time-lapse photography in cell lines carrying intact, mutated, or no EGF receptor. The G2-M transition of cells devoid of EGF receptor or carrying an EGF receptor devoid of most of the cytoplasmic domain was not influenced by EGF. In cells carrying intact EGF receptor, EGF caused a transient and dose-dependent delay in G2 phase which could last for greater than 2 h. Cells were inhibited in G2 within less than 10-20 min prior to prophase. A parasynchronous recovery from G2 inhibition was observed at large EGF concentrations; the G2-M transition rate exceeded that of the controls. The system described may represent a model for the mechanistic analysis of a ligand-induced transient restriction of the cell cycle, particularly at the G2-M border.

Animals↗

PubMed1983

[Histone synthesis at S- and G2-phases of the mitotic cycle of human diploid fibroblasts].

The phase-specific synthesis of the total protein, histones, RNA and DNA in human embryonic fibroblasts synchronized by double thymidine blockade was investigated. It was shown that activation of histone synthesis occurs in three steps, i.e. i) immediately before replication, ii) after DNA synthesis maximum, and, iii) at the G2-phase of the mitotic cycle. During maximal replication of DNA histone synthesis is suppressed. It was assumed that at the pre-replication phase histone synthesis controls decondensation, while and the G2-phase--condensation of chromatin.

Cells, Cultured↗

PubMed1996

A protein kinase-dependent block to reinitiation of DNA replication in G2 phase in mammalian cells.

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation in Xenopus egg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing.

Adenine↗

PubMed1988

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1.

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.

Animals↗

PubMed1984

Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.

Animals↗

PubMed1985

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair.

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.

Cells, Cultured↗

PubMed1975

Inhibition by 4'-demethyl-epipodophyllotoxin 9-(4,6-O-2-thenylidene-beta-D-glucopyranoside) of human lymphoblast cultures in G2 phase of the cell cycle.

4'-Demethyl-epipodophyllotoxin 9-(4,6-0-2-thenylidene-beta-D-glucopyranoside) is a semisynthetic derivative of podophyllotoxin with oncolytic activity against a variety of neoplasms, Unlike the parent compound and other derivatives, which cause methaphase arrest, 4'-Demethyl-epipodophyllotoxin 9-(4,6-0-2-thenylidene-beta-D-glucopyranoside) apparently blocks the entry of CCRF-CEM cells into mitosis by inhibiting cells in the G2 phase of the cell cycle. The inhibition of precursor incorporation into DNA, RNA, and the protein, if observed, was delayed and probably secondary to inhibition of cells in the G2 phase of the cell cycle.

Cell Count↗

PubMed1991

Differential effect of cordycepin on S and G2 phases of cell cycle in plasmodia of Physarum polycephalum Schw.

Effect of pulse treatments of cordycepin, an analog of adenosine, on S and G2 phases of the cell cycle of the mitotically synchronous plasmodia of Physarum polycephalum has been studied. Various concentrations of the drug (50-200 micrograms ml-1) were found to be effective in delaying mitosis by several hours in both the phases. However, there was a significant increase in mitotic delay in those treated during G2. It is suggested that this extra delay during G2 could be due to the transcriptive level inhibition of specific RNA types, such as that of tubulins, whose gene activity is cell cycle regulated and turned on during G2 in Physarum, or alternatively because of a deficiency for ATP and the consequent inhibition of events such as mitotic spindle assembly and phosphorylation of histones.

Animals↗