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PubMed1992

Proposed role of phosphatidic acid in the extracellular control of the transition from G2 phase to mitosis exerted by epidermal growth factor in A431 cells.

Epidermal growth factor (EGF) has been shown to cause an inhibition of A431 cells in G2 phase within approximately 10 min, i.e., shortly before mitosis (Kinzel et al., Cancer Res., 50: 7932-7936, 1990). This system has been used to study the proposed role phospholipid metabolites, particularly phosphatidic acid (PA), may play (Kaszkin et al., Cancer Res., 51: 4328-4335, 1991) in the extracellular control of cells at the physiological restriction site in G2 phase. A431 cells responded to EGF with a dose-dependent formation of phosphatidic acid (PA) which correlated with the dose-dependent G2 delay as well as with their time courses. The G2 delay induced by EGF as well as PA mobilization were effected in conditioned medium or in fresh medium containing bovine serum albimun instead of serum, i.e., under the conditions necessary for precursor studies to be carried out. The major pathway of PA formation was probably via phospholipase C-mediated breakdown of phosphatidylinositol and diacylglycerol kinase: (a) the dose response of PA formation correlated with that of total inositol phosphate accumulation; (b) little diacylglycerol was found and then only at a high EGF concentration; (c) prelabeling with [1-14C]arachidonic acid resulting in a large specific labeling of phosphatidylinositol led to an EGF-induced, dose-dependent formation of radioactive arachidonyl-PA (correlated with that of total PA and inositol phosphate), but in the presence of a primary alcohol not to the formation of radioactive phosphatidylalcohol; (d) prelabeling with [1-14C]oleic acid led to the EGF-induced formation of labeled PA, which in the presence of a primary alcohol was only slightly reduced to the advantage of very low levels of labeled phosphatidyl alcohol, thus demonstrating that an EGF-effected activation of phospholipase D did occur but contributed little to the general PA level. An alternative mobilization of PA was attempted with the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), which was shown to activate phospholipase D in A431 cells and to elicit PA from a phospholipid pool which was not significantly labeled with radioactive arachidonic acid. The TPA-induced degree of PA formation and of the G2 delay correlated. Both phenomena were considerably larger with fresh medium containing 0.5% bovine serum albumin instead of serum than in conditioned medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Drug Synergism↗

PubMed1994

Cyclin expression and G2-phase delay after irradiation.

Many studies have demonstrated the effect of oncogene transfection on the radiation sensitivity of primary rat embryo fibroblasts. Our results indicate that transformation by H-ras plus v-myc oncogenes confers radiation resistance to a much greater extent than transformation by either gene alone. We have further shown that the radioresistant phenotype is accompanied by a prolonged G2-phase delay. This is consistent with the hypothesis that the extent of this delay is an important determinant of radiation sensitivity. The study of cyclin expression during the progression of cells through G2 and M phase after irradiation has also revealed several possible mechanisms for induction of G2-phase arrest. Control of cyclin B levels was seen both at the mRNA level as evidenced by decreased cyclin B mRNA expression after irradiation in the S phase, and at the protein level as demonstrated after irradiation in G2 phase. It remains to be determined how these mechanisms might be differentially regulated in radioresistant and sensitive cells.

Animals↗

PubMed2001

Ras-dependent cell cycle commitment during G2 phase.

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

Animals↗

PubMed1995

Cell cycle progression and phenotypic modification of Ki67 antigen-negative G1- and G2-phase cells in phorbol ester-treated Molt-4 human leukemia cells.

To elucidate the relationship between the level of cellular Ki67-reactive antigen and cell proliferation, the effects of 12-O-tetra-decanoylphorbol 13-acetate (TPA) on Ki67 expression, cell cycle progression, and surface phenotypes of human T-lymphoblastic leukemia Molt-4 cells were investigated by multiparameter flow cytometry. The Ki67 antigen is constitutionally expressed in almost all untreated exponentially proliferating Molt-4 cells. Treatment with 10 nM TPA prolonged the duration of the cell cycle time and resulted in a progression arrest of cells in G1- and G2-phases, during which Ki67 expression was decreased to an undetectable level. However, in TPA-treated cultures, the Ki67-positive fraction was invariably smaller than the growth fraction as estimated from continuous 5-bromodeoxyuridine (BrdUrd) labeling curves. This discrepancy could be explained by the finding that some Ki67-negative G1 cells do not enter the resting state but instead remain in the cycling compartment. These results show that Ki67 expression of tumor cells with relatively long G1 duration is downregulated to undetectable levels in late G1-phase and the difference in the level of Ki67 expression between late G1 cells and resting G1 cells is undetectable by conventional immunological methods. Although TPA induced differentiation of Molt-4 cells into mature suppressor T cells, the phenotypic modification was not correlated with cell cycle position and Ki67 reactivity of the cells. These results suggest that growth arrest and phenotypic differentiation of Molt-4 cells are independent effects of TPA.

Antigens, CD↗

PubMed1986

Modulation in protein phosphorylation during G2 phase of the cell cycle in two-cell mouse embryos.

The protein phosphorylation activities in extracts were assayed for 2-cell mouse embryos at three stages of the G2 phase of the cell cycle. The 2-cell embryos were unique in having a prolonged G2 phase and so easily staged at early G2 (EG2), middle G2 (MG2) and late G2 (LG2) by timing the embryo isolation from pregnant mice. The embryo extracts were used both as sources of protein kinases and their substrates. The phosphoproteins of the extracts were labelled with [gamma-32P]ATP and separated by electrophoresis on SDS-polyacrylamide gels. The present study revealed that protein phosphorylation increased 3-6-fold during the progression of 2-cell embryos from EG2 to LG2 and the level of protein phosphorylation at any stages was greatly decreased by the presence of cAMP. Thus, the protein phosphorylation system of 2-cell mouse embryos seems to differ from those reported systems in mammals in its negative dependence on cAMP.

Animals↗

PubMed2004

Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells.

The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G2 than in G1, suggesting that DSB rejoining in G2 proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G1 and G2 compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G2 occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G1 and G2. We propose that structural characteristics of G2 phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.

Animals↗

PubMed1999

Chromosomal sensitivity to X-ray irradiation during the G2 phase in lymphocytes of patients with hereditary cutaneous malignant melanoma as compared to healthy controls.

Recent reports have suggested that elevated chromosomal aberration yields following X-ray irradiation of skin fibroblasts and peripheral lymphocytes in the G2 phase of the cell cycle are characteristic of affected members of cancer-prone families. These studies propose that the phenomenon is a consequence of impaired caffeine- and arabinofuranosylcytosine (ara-C)-sensitive DNA repair and might be a useful indicator of genetic susceptibility to cancer. We have tested G2 chromosomal X-ray sensitivity in peripheral blood lymphocytes from members of kindreds with hereditary cutaneous malignant melanoma (HCMM) combined with the dysplastic nevus syndrome (DNS), disorders in which susceptibility to skin cancer is inherited in an autosomal dominant pattern. In the assay lymphocytes from patients with HCMM/DNS exhibited responses indistinguishable from normal healthy controls. Furthermore, the radiation-induced aberration yields were potentiated to the same strong extent by post-treatments with caffeine, or a combination of ara-C and hydroxyurea, both in lymphocytes from individuals with HCMM/DNS and lymphocytes from healthy controls. Thus, lymphocytes of affected patients with HCMM/DNS do not have an increased sensitivity to X-ray irradiation in the G2 phase of the cell cycle.

Adult↗

PubMed1991

Prolonged G2 phase of breast cancer cells and chromosome damage.

5-bromodeoxyuridine incorporation was studied in metaphase chromosomes from 24 breast tumour specimens, including 23 adenocarcinomas. In these 23 cases, a slow cell cycle was observed, with a long (8 h) G2 phase. This slowing of the cell cycle, which was poorly related to the degree of polyploidy, was significantly related to the number of chromosome anomalies: the cell cycle was particularly slow when many rearranged chromosome were observed. These in vitro findings during the first cell cycle cannot easily be transposed to the in vivo situation. By analogy with Fanconi anaemia, in which both chromosome lesions and a long G2 phase are detected, a DNA repair defect and/or high DNA mutagenesis might exist in breast cancer cells.

Adenocarcinoma↗

PubMed1997

Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase.

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.

Animals↗

PubMedDate not supplied

Inhibition of G2 phase in unsynchronized HELA cells: synergism between adenosine 3':5'-monophosphate analogues and phosphodiesterase inhibitors.

The involvement of cyclic nucleotides of G2 phase of the cell cycle was reexamined in unsynchronized HeLa cells. Two methods were employed to determine if cell cycle traverse was affected by chemical agents. The first method analyzed cell density changes a relatively early times after drug treatment. The second method entailed selection of a cohort of "tagged" G2 cells and analysis of the progress of these cells into mitosis. Synergism between the effects of low levels of cyclic nucleotide analogues and phosphodiesterase inhibitors was used as a criterion for the specificity of an agent's action. Using both methods of cell cycle analysis, synergistic inhibition of cell division was observed with adenosine 3':5'-monophosphate analogues. No synergism was observed with various control compounds or with guanosine 3':5'-monophosphate analogues. The results are interpreted to indicate that adenosine 3':5'-monophosphate exerts an inhibitory action in G2 phase of the cell cycle.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Effect of pentoxifylline on radiation-induced G2-phase delay and radiosensitivity of human colon and cervical cancer cells.

Cells of three adherent cell lines with mutated p53 (WiDr and C33-A) and disrupted p53 (C4-I) were used to investigate the effect of pentoxifylline (PTX) on radiation-induced G2-phase block and its relationship to radiosensitivity. Postirradiation exposure to 0.25-1.0 mM PTX resulted in an increase in radiosensitivity in a concentration-dependent manner as determined by a clonogenic assay. The change in radiation sensitivity was quantified by calculating the enhancement ratio (ER) at a clinically relevant dose of 2 Gy; the ER for WiDr cells was 1.23+/-0.03 and 1.39+/-0.15 for 0.5 and 1.0 mM PTX, respectively. For C33-A cells, the ER ranged from 1.04+/-0.04 to 1.99+/-0.17 for 0.25-1.0 mM PTX, whereas for C4-I cells the values were 1.29+/-0.04 and 1.76+/-0.17 for 0.25 and 0.5 mM PTX. In asynchronous WiDr, C33-A and C4-I cells, flow cytometry analysis showed a dose-dependent accumulation of cells in G2/M phase which was detectable at 6 h and peaked at 12 h after irradiation. Such a G2/M-phase block was transient at a dose of 2 Gy and persisted at 48 or 72 h after a dose of 4 or 6 Gy. At 12 h after 2 Gy, PTX significantly reduced the radiation-induced G2/M-phase block in a dose-dependent manner. After the higher doses of 4 and 6 Gy, the dose-dependent G2-phase arrest was significantly alleviated at 24 h by treatment with PTX, and the kinetics of this alleviation depended on the radiation dose. The results demonstrate that human colon and cervical cancer cells characterized by a mutated or disrupted p53 (i.e. not transfected) are radiosensitized by PTX, which alleviates the postirradiation G2/M-phase block.

Cell Cycle↗

PubMed1995

A cell-cycle nuclear autoantigen containing WD-40 motifs expressed mainly in S and G2 phase cells.

Recently a novel nuclear protein mainly expressed in S and G2 phase cells was characterized using autoantibodies from a cancer patient (Exp. Cell Res., 212, 255-261, 1994). cDNA clones were isolated by immunoscreening, and sequence analysis revealed that the cDNA clones encoded a 713-amino acid residue polypeptide which is provisionally named SG2NA (S/G2 nuclear antigen). In the carboxyl terminal half of SG2NA, there are 6 copies of WD-40 motifs characteristic of a large family of proteins of which the prototype is beta-transducin/enhancer of split (TLE). In addition, there are nuclear localization sequence and phosphorylation sites related to the TLE proteins. Autoantibodies occurring spontaneously in certain cancer patients have been useful reagents for identifying cellular proteins and this is the first report of a novel WD-40 protein which is the target of an autoimmune response.

Adenocarcinoma↗

PubMed2006

Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCF(betatrcp).

The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCF(betatrcp). We show that Pin1 binds to Emi1 and prevents its association with betatrcp in an isomerization-dependent pathway. We also show that Emi1-Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S- and M-phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1-dependent stabilization of Emi1 during G2.

Animals↗

PubMed1988

Microtubule-dependent cell cycle regulation is implicated in the G2 phase of Hydra cells.

Interstitial cells of Hydra attenuata, from which nerve cells and nematocytes (stinging cells) differentiate, were arrested in either metaphase or G2 by different concentrations of the microtubule-depolymerizing agent nocodazole. At a concentration of 1.4 nM-nocodazole, a large number of cells were arrested in metaphase. However, at concentrations of 2 nM-nocodazole and above most of the cells were arrested at a distinct point in G2 several hours before mitosis. After removal of the 2 nM-nocodazole block, 75% of the cells entered the next cell cycle about 10 h later. To our knowledge this is the first time that cells have been synchronized by arresting them in the G2 phase. Visualization of Hydra microtubules with a tubulin monoclonal antibody and immunofluorescent staining showed that the very low concentrations of nocodazole used for cell cycle arrest were indeed affecting microtubule structures. Spindles and stem cell microtubules disappeared at 0.8-1 nM-nocodazole, followed by nerve microtubules (about 2 nM), cnidocil microtubules (10 nM) and finally by nematocyte microtubules (34 nM). Taken together, these data strongly indicate a microtubule-dependent mechanism of cell cycle regulation in the G2 phase.

Animals↗

PubMed1997

Role of wild type p53 in the G2 phase: regulation of the gamma-irradiation-induced delay and DNA repair.

Upregulation of the p53 protein was shown to induce cell cycle arrest at the G1/S border and in some cases at the G2/M border. Furthermore, it was suggested that p53 is associated with the induction of the various DNA repair pathways. Previously, we demonstrated that cells co-expressing endogenous wild type p53 protein, together with dominant negative mutant p53, exhibit deregulation of apoptosis, G1 arrest and delay in G2 following gamma-irradiation. In the present study, we investigated the role of p53 protein in the DNA damage response at the G2 phase. Using p53-null, wild type p53 and mutant p53-producer cell lines, we found that the two C-terminally spliced p53 forms could prevent gamma-irradiation induced mutagenesis prior to mitosis, at the G2/M checkpoint. We found that at the G2 phase, p53 may facilitate repair of DNA breaks giving rise to micronuclei, and regulate the exit from the G2 checkpoint. At the G1 phase, only the regularly spliced form of p53 caused growth arrest. In contrast, both the regularly and the alternatively spliced p53 forms directed postmitotic micronucleated cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in normal cycling cells, as well as in cells where p53 is up-regulated, following DNA damage.

Animals↗

PubMed2004

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM.

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.

Androstadienes↗

PubMed1996

Cytogenetic responses to G2 phase x-irradiation of cells from retinoblastoma patients.

Fibroblast cell lines from 20 retinoblastoma (RB) patients with the hereditary bilateral form of disease compared with 16 lines from normal donors had a significantly higher chromatid aberration frequency (CAF), and more displaced and nondisplaced breaks per 100 metaphase cells after x-irradiation during the G2 phase of the cell cycle. The mean CAF was 39 +/- 1.0, range 30-46, for cells from normal subjects, compared to a mean of 245.6, range 101-506, for cells from hereditary RB patients (p < 10(-6). Of fibroblast lines from eight patients with unilateral RB, four had a CAF comparable to that of lines from normal donors (< 60) and four had a high CAF (> 130), resembling that of hereditary forms; two of the latter four lines were from patients with familial or deletion 13 forms of RB. Furthermore, in two families, PHA-stimulated blood lymphocytes from RB patients, one bilateral and one unilateral, and from certain unaffected first-degree relatives after G2 phase X-irradiation had a high CAF (> or = 110) compared to a CAF (> or = 53) of cells from three normal donors sampled at the same time. These results were shown not to be related to differences in cell cycle progression or initial extent of chromatid damage. The results suggest that the high frequency of chromatid aberrations in the cells from hereditary RB patients results from a genetic deficiency in DNA repair.

Adult↗

PubMed2006

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Animals↗