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1.
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.  相似文献   

2.
20,1% cells with chromosomes aberrations were obtained after UV-irradiation of embryonal fibroblasts of mice at the S-stage in vitro at a decreasing dose of 40erg/mm2. Subsequent gamma-irradiation at the metaphase of the first mitosis at a 5 krad dose led to a statistically significant decrease of the frequency of aberrant cells observed in the same mitosis down to 11,7%. The frequency of spontaneous aberrations did not change during the first few minutes after gamma-irradiation of intact cells at the metaphase. The "protective" effect of gamma-rays can not be explained either by unequal changes of the duration of mitotic stages for aberrant and normal cells, or by sticking of chromosome fragments or by breaks of bridges at the anaphase. The death of cells "under irradiation" also appears to be a hardly probable case of the effect observed. It is assumed that the decrease of the aberrations frequency is the result of predicted earlier modification of the processes of realization of potential chromosome damages into visible aberrations at the metaphase.  相似文献   

3.
After long postirradiation incubation periods, the residual frequency of prematurely condensed chromosome fragments following X-ray exposure of noncycling diploid human fibroblasts was found to be correlated with the frequency of chromosome aberrations observed under identical treatment conditions when the cells were subcultured and scored after they reached mitosis. Over a wide range of doses, the proportion of such cells without aberrations at their first metaphase was not significantly different from the proportion able to form macroscopic colonies. Further, the rate of rejoining of interphase chromosome breaks was the same as the rate of increase in survival due to the repair of potentially lethal damage (PLD). These results suggest that there is a one-to-one correspondence between the initial breakage and rejoining of G0 chromosomes and the induction and repair of PLD measured by delayed plating from plateau-phase cultures of these cells.  相似文献   

4.
In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.  相似文献   

5.
X S Ye  R R Fincher  A Tang    S A Osmani 《The EMBO journal》1997,16(1):182-192
It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.  相似文献   

6.
Adenovirus uncouples DNA replication from polyamine biosynthesis and causes chromosome aberrations in rodent cells. Addition of polyamines protected infected cells from this chromosome damage. Spermine was the only individual polyamine which protected. The diamine oxidase inhibitor aminoguanidine also protected. Neither compound detectably reduced synthesis of viral early proteins. The protective effects of spermine and aminoguanidine were not additive. Maximal protection was obtained when the compounds were added 4.5 h before mitosis, but significant protection was observed up to 1.25 h before mitosis. This suggests that the compounds act in G2. In vitro, spermine bound strongly to DNA and protected it from mild endonuclease attack, but aminoguanidine did neither. We propose that viral infection causes a deficiency in spermine during a critical period in G2, possibly accompanied by an increase in endonuclease activity. The resulting chromosome damage can be prevented by adding exogenous spermine, or by inhibiting the oxidative degradation of endogenous spermine.  相似文献   

7.
Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.  相似文献   

8.
The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.  相似文献   

9.
The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.  相似文献   

10.
The molecular basis of drug-induced G2 arrest in mammalian cells   总被引:5,自引:0,他引:5  
Summary The purpose of this review was to focus mainly on the molecular events related to the progression of cells through the G2 period to examine the cause for G2-arrest in mammalian cells after exposure to various anticancer drugs. With few exceptions, most of the eukaryotic cells exhibit a G2 period in their life cycles. The G2 period, which separates S phase from mitosis, represents the time necessary for the synthesis of the various components related to the condensation of chromosomes, assembly of the mitotic spindle, and cytokinesis. Continued synthesis of RNA and protein is necessary for the successful completion of G2 and the initiation of mitosis. Inhibition of RNA and protein synthesis, replacement of phenylalanine by its analog parafluorophenylalanine, or the elevation of intracellular cAMP concentrations, induce reversible G2 arrest in cultured cells. Exposure of cells to certain antineoplastic drugs also blocks cells preferentially in G2. This irreversible drug-induced G2 arrest is associated with extensive chromosome damage. The G2-arrested cells were found to be deficient in certain proteins that may be specific for the G2-mitotic transition. These mitotic or chromosome condensation factors synthesized during the G2 period, reach their maximum levels at mitosis. A preliminary characterization of the chromosome condensation factor revealed that it is a heat labile, Ca2+-sensitive, nondialyzable protein with a sedimentation value of 4–5S.  相似文献   

11.
Morrison C  Rieder CL 《DNA Repair》2004,3(8-9):1133-1139
How cells behave as they divide in the presence of chromosome (DNA) damage is only just beginning to be explored. It appears to depend on the cell type and organism, the stage of development, how extensive the damage is and when it occurs. The existing data support the conclusion that vertebrate somatic cells lack a conventional DNA damage checkpoint during mitosis, and that when damaged DNA does prolong mitosis it is mediated by the spindle assembly checkpoint. As a rule, in the presence of DNA damage cells ultimately undergo an aberrant mitosis and enter the ensuing G1. They then either die, via apoptosis or mitotic catastrophe, or survive with an altered genome. To avoid these outcomes, cells with DNA damage are normally prevented from entering mitosis by a number of G2 checkpoint control pathways.  相似文献   

12.
Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.  相似文献   

13.
Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.  相似文献   

14.
Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.  相似文献   

15.
Mitotic death is a delayed response of p53 mutant tumours that are resistant to genotoxic damage. Questions surround why this response is so delayed and how its mechanisms serve a survival function. After uncoupling apoptosis from G1 and S phase arrests and adapting these checkpoints, p53 mutated tumour cells arrive at the G2 compartment where decisions regarding survival and death are made. Missed or insufficient DNA repair in G1 and S phases after severe genotoxic damage results in cells arriving in G2 with an accumulation of point mutations and chromosome breaks. Double strand breaks can be repaired by homologous recombination during G2 arrest. However, cells with excessive chromosome lesions either directly bypass the G2/M checkpoint, starting endocycles from G2 arrest, or are subsequently detected by the spindle checkpoint and present with the features of mitotic death. These complex features include apoptosis from metaphase and mitosis restitution, the latter of which can also facilitate transient endocycles, producing endopolyploid cells. The ability of cells to initiate endocycles during G2 arrest and mitosis restitution most likely reflects their similar molecular environments, with down-regulated mitosis promoting factor activity. Resulting endocycling cells have the ability to repair damaged DNA, and although mostly reproductively dead, in some cases give rise to mitotic progeny. We conclude that the features of mitotic death do not simply represent aberrations of dying cells but are indicative of a switch to amitotic modes of cell survival that may provide additional mechanisms of genotoxic resistance.  相似文献   

16.
The classical cytogenetic assay to estimate the dose to which an individual has been exposed relies on the measurement of chromosome aberrations in lymphocytes at the first post-irradiation mitosis 48 h after in vitro stimulation. However, evidence is accumulating that this protocol results in an underestimation of the cytogenetic effects of high LET radiation due to a selective delay of damaged cells. To address this issue, human lymphocytes were irradiated with C-ions (25-mm extended Bragg peak, LET: 60-85 keV/ micro m) and aberrations were measured in cells reaching the first mitosis after 48, 60, 72 and 84 h and in G2-phase cells collected after 48 h by calyculin A induced premature chromosome condensation (PCC). The results were compared with recently published data on the effects of X-rays and 200 MeV/u Fe-ions (LET: 440 keV/ micro m) on lymphocytes of the same donor (Ritter et al., 2002a). The experiments show clearly that the aberration yield rises in first-generation metaphase (M1) with culture time and that this effect increases with LET. Obviously, severely damaged cells suffer a prolonged arrest in G2. The mitotic delay has a profound effect on the RBE: RBE values estimated from the PCC data were about two times higher than those obtained by conventional metaphase analysis at 48 h. Altogether, these observations argue against the use of single sampling times to quantify high LET induced chromosomal damage in metaphase cells.  相似文献   

17.
The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells. The effect of 100 microM DL-alpha-tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5 mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample. alpha-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%). The efficiency of the G2 repair process (G2 R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2 R rate, both in FA cells and controls. These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells.  相似文献   

18.
The frequency of X-ray-induced chromosome aberrations in G1 ML-1 mouse myeloid leukemia cells and normal mouse bone marrow cells increased with post-irradiation incubation with the DNA-repair resynthesis inhibitor 1-beta-D-arabinofuranosylcytosine (araC), but the frequency of aberrations in the leukemic cells increased with quite a different time response compared to the normal cells. Irradiated normal mouse bone marrow cells had a rapid increase in the frequency of chromosome exchanges and deletions with increasing araC incubation time, for example, an increase was observed with 0.5 h araC incubation. In contrast, the ML-1 cells did not have a significant increase in aberrations until 1-2 h post-irradiation incubation with araC. These results suggest that the ML-1 cells, per unit time, initially undergo less repair of the X-ray-induced DNA damage that can be converted into chromosome aberrations. We previously showed that the ML-1 cells have a higher frequency of X-ray-induced chromosome aberrations compared to normal cells and the results presented here indicate that a slower rate of repair resynthesis is contributing to the increased sensitivity of the ML-1 cells.  相似文献   

19.
The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.  相似文献   

20.
Effects of vanillin on the induction of sister-chromatid exchanges (SCEs) and structural chromosome aberrations by mitomycin C (MMC) were investigated in cultured Chinese hamster ovary cells. Vanillin induced neither SCEs nor chromosome aberrations by itself. However, an obvious increase in the frequency of SCEs was observed when MMC-treated cells were cultured in the presence of vanillin. The effect of vanillin was S-phase-dependent. On the contrary, the frequency of cells with chromosome aberrations was significantly decreased by the post-treatment with vanillin at G2 phase.  相似文献   

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