Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of DNA double-strand breaks (DSBs] by restriction endonucleases (REs) and inhibitors of DNA topoisomerase II known to induce DNA strand breaks. Five different types of REs, namely CfoI, EcoRI, HpaII (which induce cohesive DSBs), HaeIII and AluI (which induce blunt DSBs) were employed. REs that induce blunt-end DNA DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks. xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type CHO-K1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant CHO cells, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type cells. We also studied the effect of inhibitors of DNA topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type CHO-K1 cells respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G1 treatment of CHO cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than CHO-K1 cells. The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of DNA topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells

Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the β-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of β-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.     

Gene recombination in X-ray-sensitive hamster cells.

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.     

Characterization of homologous recombination induced by replication inhibition in mammalian cells.

To analyze relationships between replication and homologous recombination in mammalian cells, we used replication inhibitors to treat mouse and hamster cell lines containing tandem repeat recombination substrates. In the first step, few double-strand breaks (DSBs) are produced, recombination is slightly increased, but cell lines defective in non-homologous end-joining (NHEJ) affected in ku86 (xrs6) or xrcc4 (XR-1) genes show enhanced sensitivity to replication inhibitors. In the second step, replication inhibition leads to coordinated kinetics of DSB accumulation, Rad51 foci formation and RAD51-dependent gene conversion stimulation. In xrs6 as well as XR-1 cell lines, Rad51 foci accumulate more rapidly compared with their respective controls. We propose that replication inhibition produces DSBs, which are first processed by the NHEJ; then, following DSB accumulation, RAD51 recombination can act.     

Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.     

Enhanced mutability at the tk locus in the radiosensitive double-strand break repair mutant xrs5

The thymidine kinase locus (tk) has been utilised as the target locus to measure the induced mutation frequency following X-irradiation in the X-ray-sensitive xrs5 mutant and its parent CHO K1 line of Chinese hamster cells. Mutations to tk- cells were measured by plating cells in selective medium containing trifluorothymidine after a post-irradiation expression time of 4 days. Our results show that the mutation frequency was 3-4 times higher in the xrs5 mutant than in the CHO K1 cell line. This enhanced mutation frequency in xrs5 is though to result from the deficiency in DNA double-strand break repair in this cell line which also results in the enhanced cell killing and higher frequencies of chromosomal aberrations in response to X-irradiation. The findings of the present study suggest that DNA double-strand break is a critical lesion leading to mutations in irradiated cells.     

Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate.

The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.     

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.     

Protection of human tumor cells of differing radiosensitivity by WR-1065

We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of gamma radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently ( approximately 1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA-drug associations.     

Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line

The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the xrs strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronied populations of xrs cells, estimated to have been irradiated in late S and G2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.     

Repair of DNA single-strand and double-strand breaks in the Chinese hamster xrs 5 mutant cell line as determined by DNA unwinding

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray-sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.