Lack of association between GSTT1 polymorphism and endogenous or benzo[a]pyrene-induced sister chromatid exchanges as analyzed in metaphase or G2-phase lymphocytes.

The objectives of the present work are (1) to verify whether genetic polymorphism in the detoxification gene GSTT1 influences the endogenous sensitivity in terms of sister chromatid exchanges (SCEs)/cell in healthy donors and (2) to test whether in vitro exposure to B[a]P in terms of SCEs/cell can be associated with polymorphism of GSTT1 gene. The presence or absence of the homozygous deletion in GSTT1 gene was determined in peripheral blood cells using multiplex-PCR. For SCEs quantitation, the cytogenetic method used thus far is based on the analysis in metaphase chromosomes. Consequently, G2-arrested cells are not included in the analysis. To overcome this shortcoming of the conventional method, we applied here SCE analysis in G2-phase prematurely condensed chromosomes (G2-PCCs) induced by calyculin-A, using a modified fluorescence-plus-Giemsa staining protocol. Compared to metaphase, a statistically significant increase in the yield of SCEs was notified in the G2-phase analysis after 48 h exposure of peripheral blood lymphocytes to 0.01-1 mM B[a]P, in both GSTT1-positive and -null donors. Therefore, the analysis of SCEs in the G2-phase using calyculin-A induced PCC methodology was shown to be more sensitive compared to the analysis at the metaphase level. Nevertheless, the results obtained do not show an association between the GSTT1 polymorphism with increased endogenous and/or B[a]P-induced SCE-frequencies in peripheral blood lymphocyte chromosomes in vitro. These results highlight not only the effect of B[a]P on cell cycle kinetics but also they demonstrate that conventional cytogenetic analysis at metaphase underestimates the cytogenetic effects of chemicals that delay cell cycle progression in G2-phase.

Investigation of bystander effects in hybrid cells by means of cell fusion and premature chromosome condensation induction.

The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by premature chromosome condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

Cytogenetic methods for biodosimetry and risk individualisation after exposure to ionising radiation.

Measurement of dicentric chromosomes in human lymphocytes has been applied to assess dose received by potentially overexposed people and estimate risk for health effects. Since the dicentrics in exposed people decrease with time, the introduction of fluorescent in situ hybridisation enables to measure stable translocations for biodosimetry and address old or long-term exposures. In addition, premature chromosome condensation, which enables analysis in interphase, offers several advantages for biodosimetry. However, dose and risk estimates derived using cytogenetics and adequate calibration curves are based on the assumption that all individuals respond equally to radiation. Since increased radiosensitivity has been associated with cancer proneness, there is particular interest for risk assessment at the individual level. Towards this end, the efficiency of dynamics that govern DNA repair and apoptosis, as well as the conserved cellular processes that have evolved to facilitate DNA damage recognition using signal transduction pathways to activate cell cycle arrest and preserve genomic integrity, are being investigated. Recent work in cancer cytogenetics and on the modulation of radiation effects at the chromosome level using changes in gene expression associated with proteins or factors such as caffeine or amifostine treatment during G(2) to M-phase transition, reconfirmed the importance of G(2) chekpoint in determining radiosensitivity and of the cdk1/cyclin-B activity in the conversion of DNA damage into chromatid breaks. G(2)-chromosomal radiosensitivity may offer, therefore, a basis for the identification or testing of key genetic targets for modulation of radiation effects and the establishment of a screening method to detect intrinsic radiosensitivity.

Constitutional pericentric inversion of chromosome 9 and hematopoietic recovery after allogeneic stem cell transplantation.

Recent reports suggest that hemopoietic stem cells with constitutional pericentric inversion of chromosome 9 [inv(9)] may be related to delayed engraftment or hemopoietic defect after stem cell transplantation (SCT). We conducted a retrospective study on five allogeneic SCT in which constitutional inv(9) was detected either in the donor or the recipient. The results showed that hematologic recovery was within the expected time range for all our patients. However, one patient exhibited decreasing blood counts between day +45 and +272 after transplantation, possibly due to protracted cytomegalovirus (CMV) infection and gansiclovir and imatinib treatment. Our findings suggest that constitutional inv(9) may not be associated with delayed hemopoietic recovery after SCT.

SCE analysis in G2 lymphocyte prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action.

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a "possible human carcinogen". The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used.

Radioprotective effect of amifostine on cells from cancer prone patients and healthy individuals studied by the G2 and PCC assays.

PURPOSE: To investigate whether amifostine is effective at reducing the yield of chromatid breaks when present during G(2)-phase irradiation of human normal cells and cells from cancer prone patients, as well as to study the mechanisms underlying the radioprotective effect of amifostine. MATERIALS AND METHODS: G(2) chromosomal radiosensitivity in the presence or absence of amifostine was studied in healthy donors, cancer patients, ataxia-telangietasia (A-T) patients and five human lymphoblastoid cell lines with genes predisposing to cancer. The yield of chromatid breaks following gamma-irradiation in G(2) phase was obtained at the subsequent metaphase using the G(2) assay. For scoring chromatid damage directly in G(2) or G(0) phase, premature chromosome condensation was used. RESULTS: When amifostine was present during irradiation, the mean yield of radiation-induced chromatid breaks as visualized by the G(2) assay was significantly reduced in healthy donors (t-test, p=0.001), in cells from cancer patients (p=0.001) and in cell lines from patients with genes predisposing to cancer (p=0.01) except ATM(-/-) (0.1

A new cytogenetic approach for the evaluation of mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase.

The aim of the present study was to develop and standardize a cytogenetic approach for evaluation of the mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase. Even though cytogenetic end-points such as sister chromatid exchange (SCE) have been extensively used to indirectly assess the DNA-damaging potential of various chemicals, they are based on metaphase chromosome analysis. Cells delayed in G2 phase after chemical exposure are not included in conventional SCE analysis. The yield of SCEs obtained, therefore, can be biased, since predominantly undamaged cells proceed to metaphase without delay. To overcome this shortcoming of conventional SCE analysis, the use of a new cytogenetic approach for genotoxic studies is presented that enables the analysis of SCEs directly in G2 phase using drug-induced premature chromosome condensation in cultured peripheral blood lymphocytes. By means of this method, firstly, the possibility that SCE analysis in metaphase chromosomes underestimates the mutagenic potential of various chemicals was tested. Secondly, whether the genotoxic potential of suspected carcinogens could be evaluated using SCE analysis in G2 phase, even at exposures that arrest cells in G2 phase, was examined. Thirdly, whether an important part of the background variation in SCE frequency among individuals is due to the delay of affected cells in G2 phase, rather than to a true biological variation in the cytogenetic end-point used, was tested. The results showed that a higher SCE frequency was scored in G2 phase than in metaphase. Subsequently, the mutagenic potential of chemicals that temporarily arrest cells in G2 phase could now be evaluated more accurately. In addition, it may be of interest to further examine the involvement of cell cycle kinetics in the baseline SCE variation among individuals since a lesser SCE variability was observed when the analysis was carried out in G2 phase rather than at metaphase.

Clonal chromosome rearrangements in hairy cell leukemia: personal experience and review of literature.

Cytogenetic studies in hairy cell leukemia (HCL) are rare. In the present report, cytogenetic investigations were performed on marrow cells obtained from 21 HCL male patients with a mean age of 57 years and active disease. Karyotypic analysis was successful in 18 of the 21 patients, either at diagnosis or in relapse after treatment with IFNa. Clonal chromosome abnormalities were detected in eight of 18 cases. The chromosome most frequently involved in the rearranged karyotypes was chromosome 14. Results are discussed with respect to 79 abnormal HCL cases obtained from an extensive review of the literature from 1978 to 2000.

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.

Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved.

PURPOSE: To test the hypothesis that deficient DNA repair as measured by increased G2 chromosomal radiosensitivity results from up-regulation of cdk1/cyclinB and cell cycle control mechanisms during the G2 to M transition. MATERIALS AND METHODS: A total of 185 cancer patients and 25 normal individuals were tested for G2 chromosomal radiosensitivity. The chromatid breaks were analysed in metaphase using the G2 assay or directly in G0 and G2 phase using premature chromosome condensation (PCC). The activity of cdk1/cyclinB, a key regulator of the G2 to M-phase transition, was measured by histone H1 kinase activity and correlated with the development of chromatid breaks after irradiation of cell lines in vitro. RESULTS: Based on the G2 assay, cancer patients on average showed increased chromosomal radiosensitivity above controls. When the analysis was carried out directly in G0 or G2 lymphocytes using PCC, no differences in the induction of chromosomal damage and its repair were observed between G2 assay-sensitive and G2-normal donors. Using the G2 assay to test G2 radiosensitivity in various cell lines, it was found that the higher the cdk1/cyclinB activity level of the cell line tested, the higher the yield of chromatid breaks scored. Furthermore, when mitotic cells from these cell lines were used for PCC induction in irradiated G2 lymphocytes it was observed that the higher the cdk1/cyclinB activity level of mitotic cells used, the higher was the induced yield of chromatid breaks. CONCLUSION: The cdk1/cyclin-B activity levels during the G2 to M transition impair DNA repair processes and play a major role in the yield of chromatid breaks induced after G2-irradiation. Regulation of cdk1/cyclinB complex activity rather than deficient repair enzymes of DNA damage may underlie the mechanisms of G2 radiosensitivity.

Trisomy 6 and double minute chromosomes in a case of chronic myelomonocytic leukemia.

A 65-year-old woman with chronic myelomonocytic leukemia was shown to have trisomy 6 and multiple double minute chromosomes. The patient had no history of prior exposure to any mutagenic or carcinogenic agents. To our knowledge, this is the first report for presence of only these two aberrations. The expression of several oncoproteins and onco-related proteins was detected immunohistochemically in bone marrow cells. Among them, only the bcl-2 oncoprotein was positively stained in 100% of myeloblasts. Although the c-myc oncogene is frequently reported to be overexpressed in myeloid disorders with double minutes and associated with chemotherapy resistance and disease aggressiveness, in our case, the c-myc oncoprotein was not positively expressed. The patient received chemotherapy and complete hematological remission was successfully achieved.

Conversion of DNA damage into chromosome damage in response to cell cycle regulation of chromatin condensation after irradiation.

Cell fusion, premature chromosome condensation (PCC) and conventional cytogenetics were used to test whether the biochemical process of chromatin condensation-decondensation throughout the cell cycle, which depends on cyclin-regulated histone H1 kinase activity, affects the conversion of DNA damage into chromosome damage and determines intrinsic cell cycle-stage radiosensitivity. Results from three sets of experiments are presented. Irradiated G0 human lymphocytes were fused to exponentially growing hamster cells and time allowed for repair, while following the hamster cells in their progress towards mitosis. Severe fragmentation was observed in the induced lymphocyte PCCs when hamster cells entered mitosis 13 h after irradiation, suggesting conversion of DNA damage into non-repairable chromosome damage during G1/S transition. When PCC was used to analyse chromosome damage directly in G0 and G2 phase lymphocytes, the induction of breaks per cell per chromatid per Gy was found to be similar, suggesting that G2 increased radiosensitivity is related to chromatin condensation occurring during G2/M transition and not to an inherent chromatin structure at this phase. When chromatin condensation-decondensation at the G1/S and G2/M transitions was modified after irradiation by using conditioned media or elevated temperature (40 degrees C), a dramatic change in the yield and the type of chromosomal aberrations was observed. All results obtained were consistent with the proposed hypothesis. They may be also helpful in the characterization of a DNA-chromosome damage conversion process which could give a biochemical explanation of the variability in radiosensitivity observed at the various stages of the cell cycle as well as among mutant cells and cells of different origin. The proposed conversion process is cell cycle-regulated and, therefore, subject to up-regulation or down-regulation following mutagen exposure and genetic alterations.

Mechanism of radiosensitization by halogenated pyrimidines: the contribution of excess DNA and chromosome damage in BrdU radiosensitization may be minimal in plateau-phase cells.

We measured the contribution of increased DNA double-strand break (dsb) and interphase chromosome break induction in BrdU-mediated radiosensitization in exponentially growing (DNA dsb measurements only) and plateau-phase Chinese hamster ovary cells using an approach developed by Webb et al. (1993). The approach is based on the scavenging capacity of acetone for hydrated electrons, which are thought to react with bromine and form excess DNA and chromosome damage in BrdU-containing cells. In irradiated exponentially growing cells, acetone (1 M) removes the majority of excess DNA dsb induced in the presence of 4 microM BrdU (approximately 40% replacement of thymidine by BrdU), but does not restore cell radiosensitivity to the levels observed in BrdU-free cells. Although BrdU radiosensitizes cells by decreasing both D0 and Dq of the survival curve, acetone only restores D0 to levels measured in BrdU-free cells, but leaves Dq at levels measured in BrdU-containing cells. In plateau-phase cells, acetone removes the majority of excess DNA dsb and interphase chromosome breaks induced in the presence of 4 microM BrdU (approximately 50% replacement of thymidine by BrdU) but has only a small effect on BrdU-mediated radiosensitization to killing. These observations suggest that increased DNA damage production has a variable contribution in BrdU radiosensitization: it constitutes a major, albeit not the sole, component in the radiosensitization of exponentially growing cells, but only a minor component in the radiosensitization of plateau-phase cells. The results suggest that BrdU radiosensitization does not derive exclusively from increased DNA damage induction and support our previous hypothesis invoking repair inhibition/damage fixation as a component in the mechanism of radiosensitization. The results further suggest that repair inhibition is a major component in BrdU radiosensitization in exponentially growing cells, but the main cause of radiosensitization in plateau-phase cells.

Mitosis-promoting factor activity of inducer mitotic cells may affect radiation yield of interphase chromosome breaks in the premature chromosome condensation assay.

We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of premature chromosome condensation to study the yield and the repair kinetics of radiation damage in interphase chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of premature chromosome condensation, uncovered two times more interphase chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to chromosome breaks, and subsequently to other types of lethal chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.

Increased frequency of formation of interphase ring-chromosomes in radiosensitive irs-1 cells exposed to X-rays.

Induction and rejoining of interphase chromosome breaks were measured, after exposure to X-rays, in plateau-phase Chinese hamster V79 cells and in a radiosensitive mutant cell line derived from them, iris-1, using premature chromosome condensation (PCC). There was no difference in the induction of interphase chromosome breaks per Gy between the radiosensitive mutant cells and wild-type V79 cells despite the large differences in their radiosensitivity; an induction of 2.85 +/- 0.05 breaks/cell/Gy was measured in both cell lines. Also, rejoining of interphase chromosome breaks proceeded in the two cell lines with similar kinetics (t1/2 = 26.2 min). In contrast, ring chromosome formation was higher in irs-1 cells, as compared to wild-type V79 cells (0.78 versus 0.44 after 6 h of repair). These results confirm previous observations suggesting that a general deficiency in the rejoining of DNA dsb is unlikely to be a direct cause of the increased radiosensitivity of irs-1 cells, and are consistent with the hypothesis that the increased radiosensitivity of these cells derives from an increase in the probability of misrepair.

Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes.

Induction of premature chromosome condensation enables direct observation of radiation-induced cytogenetic damage in non-stimulated, interphase, human peripheral blood lymphocytes. This phenomenon can be explored in radiation protection for biological dosimetry in instances of accidental exposure to ionizing radiation. Quantification of an exposure by means of this approach has been limited so far mainly to the analysis of chromosome fragments. This limitation is due to the fact that conventional Giemsa staining of prematurely condensed chromosomes (PCCs) does not allow visualization of the centromeric regions and, as a result, the identification of dicentrics, centric rings and acentric fragments. In the present report a C-banding procedure, refined to avoid swelling and chromosome distortion of freshly prepared PCCs spreads, is used to identify such aberrations in non-stimulated human lymphocytes. The method allows immediate banding of the centromeric regions and enables scoring of aberrations within a time interval (3-4 h after blood sample withdrawal) that is only a fraction of that normally required when cells stimulated to proliferate are analysed at metaphase. The dose-response for dicentrics and centric rings measured in interphase lymphocytes was found to be similar to that obtained at metaphase. Measurement of dicentrics and centric rings in prematurely condensed chromosomes of human lymphocytes would provide valuable information on radiation dose estimates, especially in cases of extreme urgency.

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Production and repair of chromosome damage in an X-ray sensitive CHO mutant visualized and analysed in interphase using the technique of premature chromosome condensation.

Production and repair of chromosome damage were studied in interphase xrs-5 cells by means of premature chromosome condensation (PCC). The results obtained were compared with those previously reported for CHO cells. Production of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2.6 than in CHO cells (5.2 breaks per cell per Gy). Changes in chromatin structure, associated with the radiation-sensitive phenotype of xrs-5 cells, that increase the probability of conversion of a DNA double-stand break (dsb) to a chromosome break are involved to explain this effect. Repair of chromosome breaks as measured in plateau-phase G1 cells was deficient in xrs-5 cells and the number of residual chromosome breaks was practically identical to the number of lethal lesions calculated from survival data. This observation suggests that non-repaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of three in xrs-5 compared with CHO cells. This increase in ring formation suggests an increase in the probability of misrepair of chromosome damage that may stem either from the reduced ability of xrs-5 cells to repair dsb, or from the higher production of chromosome fragments observed per cell and per Gy.