Control of the G2/M checkpoints after exposure to low doses of ionising radiation: Implications for hyper-radiosensitivity

Two molecularly distinct G2/M cell cycle arrests are induced after exposure to ionising radiation (IR) depending on the cell cycle compartment in which the cells are irradiated. The aims of this study were to determine whether there are threshold doses for their activation and investigate the molecular pathways and possible links between the G2 to M transition and hyper-radiosensitivity (HRS). Two human glioblastoma cell lines (T98G–HRS⁺ and U373–HRS^?) unsynchronized or enriched in G2 were irradiated and flow cytometry with BrdU or histone H3 phosphorylation analysis used to assess cell cycle progression and a clonogenic assay to measure radiation survival. The involvement of ATM, Wee1 and PARP was studied using chemical inhibitors. We found that cells irradiated in either the G1 or S phase of the cell cycle transiently accumulate in G2 in a dose-dependent manner after exposure to doses as low as 0.2 Gy. Only Wee1 inhibition reduced this G2 accumulation. A block of the G2 to M transition was found after irradiation in G2 but occurs only above a threshold dose, which is cell line dependent, and requires ATM activity after exposure to doses above 0.5 Gy. A failure to activate this early G2/M checkpoint correlates with low dose radiosensitization. These results provide evidence that after exposure to low doses of IR two distinct G2/M checkpoints are activated, each in a dose-dependent manner, with distinct threshold doses and involving different damage signalling pathways and confirm links between the early G2/M checkpoint and hyper-radiosensitivity.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m² UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G₁/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m² UVB during G₀/G₁ phase had a higher survival rate than the cells irradiated during S/G₂/M phase. A minority of cells could escape S/G₂/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G₁/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m² resulted in a greater number of apoptotic cells.     

Variations in Several Responses of HeLa Cells to X-Irradiation during the Division Cycle

Several responses of synchronized populations of HeLa S3 cells were measured after irradiation with 220 kev x-rays at selected times during the division cycle. (1) Survival (colony-forming ability) is maximal when cells are irradiated in the early post-mitotic (G₁) and the pre-mitotic (G₂) phases of the cycle, and minimal in the mitotic (M) and late G₁ or early DNA synthetic (S) phases. (2) Markedly different growth patterns result from irradiation in different phases: (a) Prolongation of interphase (division delay) is minimal when cells are irradiated early in G₁ and rises progressively through the remainder of the cycle. (b) Cells irradiated while in mitosis are not delayed in that division, but the succeeding division is delayed. (c) Persistence of cells as metabolizing entities does not depend on the phase of the division cycle in which they are irradiated. (3) Characteristic perturbations of the normal DNA synthetic cycle occur: (a) Cells irradiated in M suffer a small delay in the onset of S, a slight prolongation of S, and a slight depression in the rate of DNA synthesis; the major delay occurs in G₂. (b) Cells irradiated in G₁ show no delay in the onset of S, and essentially no alteration in the duration or rate of DNA synthesis; G₂ delay is minimal. (c) Cells irradiated in S suffer an appreciable S prolongation and a decreased rate of DNA synthesis; G₂ delay is shorter than S delay.     

Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Three independent mechanisms for arrest in G2 after ionizing radiation

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G₂-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G₂-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G₂ phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2hours after IR (0.5 or 4Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6hours after IR) of cells that were in G₂/S/G₁ at the time of irradiation (4Gy) was inactivated by caffeine. This late G₂-arrest could not be explained by down-regulation of genes with functions in G₂/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1–depleted cells. TP53 was not necessary for the late G₂-arrest, but mediated an intermediate arrest (2-10hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G₂ is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.     

Effect of gamma irradiation on nucleolar activity in root tip cells of tetraploid Triticum turgidum ssp. durum L

Ionizing irradiation induces positive or negative changes in plant growth (M1) depending on the amount of irradiation applied to seeds or plant parts. The effect of 50–350 Gy gamma irradiation of kernels on nucleolar activity, as an indicator of metabolic activity, in root tip cells of tetraploid wheat Triticum turgidum ssp. durum L. cv. Orania (AABB) was investigated. The number of nucleoli present in nuclei and micronuclei as well as the mitotic index in the different irradiation dosages was used as an indicator of the cells entering mitosis, the chromosomes with nucleolar organizer regions that are active as well as chromosome doubling in the event of unsuccessful mitotic division. Nucleolar activity was investigated from 17.5 to 47.5 h after the onset of imbibition to study the first mitotic division and its consequences on the cells that were in G₂ and G₁ phases at the time of gamma irradiation. Untreated material produced a maximum of four nucleoli formed by the nucleolar organizing regions (NORs) on chromosomes 1B and 6B. In irradiated material, additional nucleoli were noted that are due to the activation of the NORs on chromosome 1A in micronuclei. The onset of mitosis was highly significantly retarded in comparison to the control due to checkpoints in the G₂ phase for the repairing of damaged DNA. This study is the first to report on the appearance of nucleoli in micronuclei as well as activation of NORs in the micronuclei that are inactive in the nucleus and the effect of chromosome doubling on nucleolar activity in the event of unsuccessful mitotic division.

     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G₂, i.e. the fraction of BrdUrd-negative G₂ cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G₂ and in S are equally delayed. S phase transit of cells irradiated in S or in G₁ did not appear to be affected. There were apparent changes in flow of cells out of G₁, which could be explained by the delayed entry of G₂ cells into the compartment because of G₂ arrest. Thus, in asynchronous cells the method was able to detect G₂ delay in those cells irradiated in S and G₂ phases and demonstrate the absence of cell-cycle delays in other phases.     

Analysis of radiation‐induced changes to human melanoma cultures using a mathematical model

Objectives: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. Materials and methods: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G₁‐, S‐ and G₂/M‐phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. Results: The model demonstrated that irradiation at 9 Gy induced G₂‐phase arrest in all lines for at least 96 h. Two cell lines with wild‐type p53 status additionally exhibited G₁‐phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G₁/S and G₂/M‐phase transitions, was broadly comparable with results of clonogenic assays. Conclusions: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long‐term cell cycle arrest.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging

Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G₂/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G₂/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G₂/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.     

Induced expression of the IER5 gene by γ-ray irradiation and its involvement in cell cycle checkpoint control and survival

The immediate-early response gene 5 (IER5) was previously shown, using microarray analysis, to be upregulated by ionizing radiation. Here we further characterized the dose- and time-dependency of radiation-induced expression of IER5 at doses from 0.5 to 15 Gy by quantitative real-time PCR analyses in HeLa cells and human lymphoblastoid AHH-1 cells. A radiation-induced increase in the IER5 mRNA level was evident 2 h after irradiation with 2 Gy in both cell lines. In AHH-1 cells the expression reached a peak at 4 h and then quickly returned to the control level, while in HeLa cells the expression only remained increased for a short period of time at around 2 h after irradiation before returning to the control. After high-dose irradiation (10 Gy), the induction of the IER5 expression was lower and delayed in AHH-1 cells as compared with 2-Gy irradiated cells. In HeLa cells, at this dose, two peaks of increased expression were observed 2 h and 12–24 h post-irradiation, respectively. RNA interference technology was employed to silence the IER5 gene in HeLa cells. siRNA-mediated suppression of IER5 resulted in an increased proliferation of HeLa cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of HeLa cells to radiation doses of up to 6 Gy, but barely affected the sensitivity of cells at 8 Gy. Moreover, suppression of IER5 potentiated radiation-induced arrest at the G2-M transition and led to an increase in the fraction of S phase cells. Taken together, we propose that the early radiation-induced expression of IER5 affects the radiosensitivity via disturbing radiation-induced cell cycle checkpoints.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

Cycle delay in irradiated cytochalasin-induced polykaryons. Does cytoskeleton status affect cell cycle checkpoints?

Following exposure of CHO-K1 cells to¹³⁷Cs irradiation at doses up to 20Gy, a delay in G₂ was observed to occur in cells permitted to divide normally, while cells induced to become giants by means of cytochalasin B demonstrated a minimal delay in the transition 2C–8C suggesting that the inhibition of cytokinesis results in modification of one or more cell cycle checkpoints. We postulate that this may occur as a consequence of damage tolerance, or by a feedback loop resulting from the reorganisation of the cytoskeleton that precludes cytokinesis.     

MAPK Pathway Activation Delays G2/M Progression by Destabilizing Cdc25B

Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G₂ phase delays entry into mitosis; however, the role of the MAPK pathway during G₂/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G₂ phase delay independent of known G₂ phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G₂ phase checkpoint arrest. Both the G₂ phase delay and blocked exit from the G₂ checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G₂/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G₂ phase delay. Thus, we have demonstrated a new function for MEK1 that controls G₂/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G₂ phase checkpoint arrest.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.