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.     

Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Effectiveness of 1.5 keV aluminium K and 0.3 keV carbon K characteristic X-rays at inducing DNA double-strand breaks in yeast cells

Induction of DNA double-strand breaks in diploid wild-type yeast cells, and inactivation of diploid mutant cells (rad54-3) unable to repair DNA double-strand breaks, were studied with aluminium K (1.5 keV) and carbon K (0.278 keV) characteristic X-rays. The induction of DNA double-strand breaks was found to increase linearly with absorbed dose for both characteristic X-rays. Carbon K X-rays were more effective than aluminium K X-rays. Relative to 60Co gamma-rays the r.b.e.-values for the induction of DNA double-strand breaks were found to be 3.8 and 2.2 for carbon K and aluminium K X-rays respectively. The survival curves of the rad54-3 mutant cells were exponential for both ultrasoft X-rays. For inactivation of rad54-3 mutant cells, the r.b.e.-values relative to 60Co gamma-rays were 2.6 and 2.4 for carbon K and aluminium K X-rays, respectively. The DNA double-strand break data obtained with aluminium K and carbon K X-rays are in agreement with the data obtained for gene mutation, chromosome aberrations and inactivation of mammalian cells, suggesting that DNA double-strand breaks are the possible molecular lesions leading to these effects.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

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.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

A comparison of aberration distribution and cell-cycle progression in cells treated with bleomycin with those exposed to X-rays

The extent of cell-cycle delay and the frequency of aberrant metaphases induced by bleomycin (BLM) and X-rays have been compared at doses which produce similar frequencies of chromosome aberrations by the 2 clastogenic agents (BLM, 40 micrograms/ml and X-rays, 2 Gy) in muntjac lymphocytes. The frequency of aberrant metaphases was low in BLM-treated cells; however, the number of aberrations per metaphase was higher than in cells exposed to X-rays. Thus in contrast to their uniform sensitivity to X-rays, the lymphocytes showed differential sensitivity to BLM. This might be due to differences among the cells in their uptake of BLM and/or its action on the nuclear membrane-DNA complex. In spite of the total number of chromosome aberrations being similar to that induced by X-rays, BLM did not induce a significant delay in cell-cycle progression as observed in the case of X-rays. A possible explanation could be that the DNA damages being limited to fewer cells than in the case of X-irradiation, the BLM-treated cultures had more normal cells allowing faster progression and/or unlike X-rays BLM may not be causing other cellular damages in addition to DNA breaks.     

Changes in the nuclear structure in the radiation-sensitive CHO mutant cell, xrs-5

The structural organization of the cell nucleus was investigated by transmission electron microscopy in the radiosensitive Chinese hamster ovary (CHO) cell mutant, xrs-5 (D0 = 45 cGy), relative to parental K1 cells (D0 = 200 cGy). In 99% of all xrs-5 cells, the outer layer of the nuclear envelope was separated from the inner layer, while 96% of K1 cells had closely apposed layers. This separation of the inner and outer layers of the nuclear envelope in xrs-5 cells was not explained by an increased susceptibility of xrs-5 cells to osmotically induced changes because (1) xrs-5 cells retained the altered nuclear periphery even when several different fixation protocols were used and (2) xrs-5 cells were not more susceptible to cell lysis as measured by trypan blue dye exclusion or by the extracellular presence of lactate dehydrogenase. The difference in the morphological organization in the nuclear periphery of xrs-5 cells correlated with the radiation sensitivity of the cells; xrs-5 cells which spontaneously reverted to a radiation sensitivity similar to that of K1 cells also reverted to a nuclear morphology similar to that of K1 cells. The inner and outer layers of the nuclear envelope were retained in nuclear scaffolds isolated from K1 and xrs-5 cells, indicating that components of the nuclear periphery are part of the nuclear scaffold. These data show that xrs-5 cells have an altered nuclear periphery which correlates with the radiation sensitivity of the cells. The separation of the layers of the nuclear envelope may represent an altered template for repair of DNA damage at the nuclear scaffold and thus may play a role in the defective repair of X-ray-induced DNA double-strand breaks in xrs-5 cells.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.     

Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells

Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations.

In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor.

The significance of these results is discussed.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on cell killing and interphase chromosome breakage in radiation-sensitive cells

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of chromosome damage by radiation was studied in plateau-phase xrs-5 cells using the premature chromosome condensation (PCC) method. It is well known that xrs-5 cells are sensitive to ionizing radiation and defective in the repair of radiation-induced DNA double-strand breaks, chromosome damage, and potentially lethal damage (PLD). Compared to repair-proficient CHO 10B cells, a reduction was observed in the overall BrdU-mediated radiosensitization in plateau-phase xrs-5 cells for the same degree of thymidine replacement. This finding is interpreted with a model for BrdU-induced radiosensitization advanced previously, in which two distinct components act to produce the overall radiosensitization observed. One component involves processes associated with the increase in initial damage (DNA and chromosome) production per unit absorbed dose and causes an increase in the slope of the survival curve, while the second component involves enhanced fixation of radiation-induced damage (PLD) and causes a reduction in the width of the shoulder of the survival curve. It is suggested that in plateau-phase xrs-5 cells, the deficiency in the repair of radiation-induced damage compromises BrdU-mediated radiosensitization by leaving active only the radiosensitization component that is associated with an increase in damage induction. Enhancement of cell killing by BrdU in plateau-phase xrs-5 cells resulted in a decrease in D0, the relative value of which was similar to the relative increase in the production of chromosome damage as measured by the PCC method. The relative values for the change in D0 and the production of chromosome aberrations were similar in plateau-phase CHO 10B and xrs-5 cells, suggesting that the physicochemical and/or biochemical processes associated with this phenomenon are the same in the two cell lines. Radiosensitization of a magnitude similar to that observed in exponentially growing CHO 10B cells was induced by BrdU in exponentially growing xrs-5 cells. This effect is attributed to a partial expression of the repair gene (transiently during S phase in all cells, or throughout the cycle in a fraction of cells) that permits some repair of radiation-induced damage and which is compromised by BrdU.     

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G₁ and G₂ stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G₂ cells, whereas in G₁ cells an increase in cromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G₂ cells following X-irradiation and the influence of different parts of the G₂ stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G₁ cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G₁ has been assumed to be due to conversion of some of the damaged bases strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.     

Evidence that DNA double-strand breaks initiate the phenotype of delayed reproductive death in Chinese hamster ovary cells.

A persistently reduced cloning efficiency occurs in many of the cloned progeny of Chinese hamster ovary (CHO) cells surviving X irradiation, a stable phenotype we have previously termed delayed reproductive death (Int. J. Radiat. Biol. 60, 483-496, 1991). We now report that this phenotype is also induced by the alkylating agent ethyl methanesulfonate (EMS), but not by irradiation with ultraviolet light. The restriction endonuclease HinfI, which binds at G [symbol: see text] ANTC DNA sequences and generates cohesive-end double-strand breaks, was also efficient in inducing delayed reproductive death. On the other hand, an X-ray-sensitive CHO mutant, xrs-5, which is defective in the repair of DNA double-strand breaks, did not show this phenotype following X irradiation. These results suggest that DNA double-strand breaks, as well as the endogenous repair processes for these breaks, are associated with the induction of the delayed reproductive death phenotype in CHO cells. The possible mechanism for the induction of delayed reproductive death by EMS is discussed.     

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.     

Relative biological effectiveness of fission neutrons for induction of micronucleus formation in mouse reticulocytes in vivo

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.     

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.     

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.     

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.