Reduction of radiation-induced cell cycle blocks by caffeine does not necessarily lead to increased cell killing

The effect of caffeine upon the radiosensitivities of three human tumor lines was examined and correlated with its action upon the radiation-induced S-phase and G2-phase blocks. Caffeine was found to reduce at least partially the S-phase and G2-phase blocks in all the cell lines examined but potentiated cytotoxicity in only one of the three tumor lines. That reductions have been demonstrated to occur in the absence of increased cell killing provides supporting evidence for the hypothesis that reductions may not be causal in those cases when potentiation of radiation-induced cytotoxicity is observed in the presence of caffeine.     

Comparative effects of caffeine on radiation- and heat-induced alterations in cell cycle progression

The effects of 3 mM caffeine on cell cycle progression of HeLa S3 cells exponentially and asynchronously growing in suspension culture were studied following exposure to 6.8 Gy gamma irradiation or 30 min at 45 degrees C hyperthermia. The stathmokinetic method, in which cells are grown in the presence of colcemid for the duration of experiment, in combination with two flow cytometric techniques, propidium iodide staining of DNA and acridine orange staining following acid denaturation of chromatin, were used to determine the fraction of cells in four cell cycle compartments, G1, S, G2, and M. Radiation and caffeine acted in a complementary manner, in which radiation reduced the caffeine-induced delays in cell cycle progression and caffeine prevented completely the radiation-induced accumulation of cells in G2 and mitotic delay. Heat and caffeine had additive effects on alterations in cell cycle progression. Cells containing spontaneous prematurely condensed chromatin were observed transiently immediately following heat exposure. These cells appeared to be in G2 and late S phase.     

Caffeine induces TP53-independent G(1)-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner

Caffeine is a model radiosensitizing agent that is thought to work by abrogating the radiation-induced G(2)-phase checkpoint. In this study, we examined the effect that various concentrations of caffeine had on cell cycle checkpoints and apoptosis in cells of a human lung carcinoma cell line and found that a concentration of 0.5 mM caffeine could abrogate the G(2)-phase arrest normally seen after exposure to ionizing radiation. Surprisingly, at a concentration of 5 mM, caffeine not only induced apoptosis by itself and acted synergistically to enhance radiation-induced apoptosis, but also induced a TP53-independent G(1)-phase arrest. Examination of the molecular mechanisms by which caffeine produced these effects revealed that caffeine had opposing effects on different cyclin-dependent kinases. CDK2 activity was suppressed by caffeine, whereas activity of CDC2 was enhanced by suppressing phosphorylation on Tyr15 and by interfering with 14-3-3 binding to CDC25C. These data indicate that the effect of caffeine on cell cycle checkpoints and apoptosis is dependent on dose and that caffeine acts through differential regulation of cyclin-dependent kinase activity.     

Effects of ionizing radiation on cell cycle progression

Irradiation of normal eukaryotic cells results in delayed progression through the G1, S, and G2 phases of the cell cycle. The G1 arrest is regulated by the p53 tumor suppressor gene product. Irradiation results in increased expression of p53, which in turn induces a 21 kDa protein, WAF 1/Cip 1, that inhibits cyclin CDK kinases. S-phase delay is observed after relatively high doses of radiation. This delay has both radiosensitive and radioresistant components, corresponding to inhibition of DNA replicon initiation and DNA chain elongation, respectively. The mechanism for this delay is as yet undefined, but the extent of the delay appears to be under genetic control and is sensitive to the kinase inhibitor staurosporine. A delay in G2 has been demonstrated in virtually all eukaryotic cells examined in response to irradiation. Our studies have focused on the mechanisms responsible for this delay. Cyclin B1 and p34^cdc2 are cell cycle control proteins that together form a kinase complex required for passage through G2 and mitosis [22]. Control of radiation-induced G2 delay is likely therefore to involve modulation of cyclin B1/p34^cdc2 activity. We have shown in HeLa cells that cyclin B1 expression is decreased in a dose-dependent manner following irradiation. This decrease is controlled at both the level of mRNA and protein accumulation. We have also shown that radiation-sensitive rat embryo fibroblast lines (REF) immortalized with v- or c-myc display a minimal G2 delay when compared to radiation resistant cells transformed with v-myc + H-ras. These REF lines respond to irradiation with a decrease in cyclin B mRNA, which parallels the extent of their respective G2 delays. The duration of the G2 delay in radiation-resistant REF can be shortened by treatment with low doses of the kinase inhibitor staurosporine. We have also been able to markedly reduce the radiation-induced G2 delay in HeLa cells using either staurosporine or caffeine. Attenuation of the G2 delay is accompanied by reversal of the radiation-induced inhibition of cyclin B mRNA accumulation. The results of these studies are consistent with the hypothesis that reduced expression of cyclin B in response to radiation is in part responsible for the G2 delay. The duration of the G2 delay may also be influenced by the activation state of the cyclin B/p34_cdc2 complex.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Caffeine could not efficiently sensitize homologous recombination repair-deficient cells to ionizing radiation-induced killing

Caffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells. Radiation-induced DNA double-strand breaks (DSBs) are repaired by two major processes, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). It remains unclear which repair process, HRR or NHEJ, is affected when the checkpoint responses are abolished by caffeine. In this study we observed the effect of caffeine on gene-targeted DT40 chicken lymphoblast cells. We show that caffeine efficiently abolishes S- and G(2)-phase checkpoint responses after irradiation in all cell lines tested and greatly radiosensitizes wild-type and ATM(-/-) cells, the partially checkpoint-deficient cells. However, caffeine has a much smaller radiosensitizing effect on RAD54(-/-) cells and has no effect on RAD51-deficient cells. RAD51 and RAD54 are the important factors for HRR. Our results indicate that the checkpoint responses abolished by caffeine (S and G(2)) mainly affect HRR, which results in cell radiosensitization.     

Radiation-induced progression delay in HeLa cells blocked in S phase by aphidicolin

The duration of the cell cycle in synchronous cultures of HeLa S3 cells that were either irradiated with 3.5 Gy of 220-kV X rays in mid-S phase or treated in early G1 or mid-S phase for several hours with 1 or 3 microM aphidicolin, or were subjected to both treatments, was measured by time-lapse cinemicrography. When compared with the generation time of untreated cells, the delay in cell progression with the combined treatment was found to be less than the sum of the delays with the individual treatments, but longer than the imposed delay caused by treatment with aphidicolin alone. Because recovery from potentially lethal radiation damage proceeds in the presence of aphidicolin, this finding suggests that a portion of the radiation-induced delay in cell progression may be associated with processes other than those that directly affect cell viability. It was also observed that the incidence of both spontaneous and radiation-induced sister-cell fusion is decreased in cultures incubated in the presence of aphidicolin.     

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X- irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine

We have studied two X-ray-sensitive mutants xrs 5 and xrs 6 (derived from the CHO-K1 cell line), known to be defective in repair of double-strand breaks, for cell killing and frequency of the chromosomal aberrations induced by X-irradiation. The survival experiments showed that mutants are very sensitive to X-rays, the D0, for the wild-type CHO-K1 was 6-fold higher than D0 value for the mutants. The modal number of chromosomes (2 n = 23) and the frequency of spontaneously occurring chromosomal aberrations were similar in all 3 cell lines. X-Irradiation of synchronized mutant cells in G1-phase significantly induced both chromosome- and chromatid-type of aberrations. The frequency of aberrations in xrs mutants was 12-fold more than in the wild-type CHO-K1 cells. X-Irradiation of G2-phase cells also yielded higher frequency of aberrations in the mutants, namely 7-8-fold in xrs 5 and about 3.5-fold in xrs 6 compared to the wild-type CHO-K1 cells. There was a good correlation between relative inability to repair of DNA double-strand breaks and induction of aberrations. The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase on the frequency of X-ray-induced chromosomal aberrations in these 3 cell lines was also studied. 3AB potentiated the frequency of aberrations in G1 and G2 in all the cell types. In the mutants, 3AB had a potentiating effect on the frequency of X-ray-induced chromosomal aberrations only at low doses. X-Ray-induced G2 arrest and its release by caffeine was studied by cytofluorometric methods. The relative speed with which irradiated S-G2 cells progressed into mitosis in the presence of caffeine was CHO-K1 greater than xrs 5 greater than xrs 6. Caffeine could counteract G2 delay induced by X-rays in CHO-K1 and xrs 5 but not in xrs 6. Large differences in potentiation by caffeine were observed among these cells subjected to X-rays and caffeine post-treatment for different durations. These responses and possible reasons for the increased radiosensitivity of xrs mutants are discussed and compared to ataxia telangiectasia (A-T) cells and a radiosensitive mutant mouse lymphoma cell line.     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

Role of arachidonic acid release in the G2 delay induced by tumor promoter TPA in HeLa cells

The tumor promoter TPA2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells (V. Kinzel, J. Richards, and M. St?hr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (greater than 10(-4) M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetylglycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.     

Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt2cAMP. Treatment of H35 cells with cAMP or Bt2cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and a depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt2cAMP on radiation-induced cell death was studied during fractionated irradiation with 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt2cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that whereas cAMP had no effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.     

Cohesin promotes the repair of ionizing radiation-induced DNA double-strand breaks in replicated chromatin

The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human cells in the G2 phase of the cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by γH2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase cells in an ATM-dependent pathway.     

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.     

A comparative study of the potentiating effect of caffeine and poly-D-lysine on chromosome damage induced by X-rays in plant cells.

X-Ray-induced chromosomal aberrations (CA) were potentiated by post-treatments in G2 with either caffeine (caff) or poly-D-lysine (PDL) in root-tip cells of Allium cepa. The enhancement of the yield of CA was concomitant with an increase in the frequency of mitosis. Our results seem to support the idea of a direct relationship between radiation-induced G2 delay and repair of chromosome damage. Here we report on similarities between caffeine and PDL in both decreasing G2 delay and enhancing chromatid aberration yield. The possible molecular mechanism(s) of action responsible for the cytogenetic effects observed are discussed.     

The relationship between radiosensitivity and cell kinetic effects after low- and high-dose-rate irradiation in five human tumors in nude mice

Radiation-induced synchronization of cells in the radiosensitive G2 phase can, theoretically, be applied to individual tailoring of fractionation schemes, possibly rendering radiotherapy more effective. For that purpose, cell cycle perturbations were studied in five xenografts by flow cytometry. A dose-dependent increase of cells in G2 phase was noticed in all five tumor cell lines after high-dose-rate irradiation, and in four tumor cell lines after low-dose-rate irradiation. The timing of maximum accumulation was not related to dose, but coincided with the cell cycle time of the respective tumors. Furthermore, the increase in the number of cells in G2 phase correlated with the radiosensitivity of the tumors as assessed by measurements of regrowth delays. The observed synchronization provides a basis for further investigations on the relevance of radiation-induced cell cycle synchrony to the effectiveness of fractionated radiotherapy.     

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.     

Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 micrograms/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 micrograms/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 micrograms/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 micrograms/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.