Dag Jenssen

Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells

ABSTRACT Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks.

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.

The micronucleus test as part of a short-term mutagenicity test program for the prediction of carcinogenicity evaluated by 143 agents tested.

To evaluate the usefulness of the micronucleus test as a short-term assay for the detection of carcinogens, the correlation between micronucleus test data for 143 chemicals and corresponding cancer data, has been analyzed. For comparison, analogous data from Amess test have also been collected for the same chemicals. In a comparison of the micronucleus test and Amess test it was found that they had about the same specificity (around 80%) and predictive value (around 90%), while there was a significant difference in sensitivity in favor of Amess test. The difference in sensitivity could be partly explained by differences in metabolizing capacities of these two test systems. It is concluded that a more elaborate test procedure for the micronucleus test would increase that sensitivity of this test. The principal value of the micronucleus test lies in the fact that it is an in vivo method, which may pick up effects at the chromosomal level not covered by bacterial assays. This is emphasized by the finding that the combination of Amess test and the micronucleus test did increase the sensitivity of the screening procedure for the prediction of carcinogenic effects.

Dose response at low doses of x-irradiation and mms on the induction of micronuclei in mouse erythroblasts.

The test of induced micronuclei in erythrocytes of mammalian bone marrow constitutes, because of its high experimental resolution power, a suitable method for the screening of induced chromosomal lesions at very low dosages of chemicals or irradiations. This test was used for a comparative investigation of the effect of low dose levels of X-irradiation and of the alkylating agent methyl methanesulphonate (MMS). The dose-effect curve of X-irradiation indicated a deviation from linearity at 10 rad, showing a significantly stronger effect than expected on extrapolation from the control to 100 rad. This deviation from linarity, however, only appeared at a low dose rate (18 R/min), whereas a linear dose-effect relation was indicated with a high dose rate (95 R/min). Experiments at 10 rad with different dose rates at two different current potentials suggested that this effect of the dose rate is more pronounced with soft than with hard X-irradiation. The induction of micronuclei with MMS follows a drastically different dose-effect curve as compared with X-irradiation. The relative efficiency of the treatment is lowest at low concentrations, presumably as a result of the efficient repair process at such dose levels. Simultaneous treatment with X-rays and MMS at low dose levels only resulted in an additive effect. This suggests that X-irradiation does not interfere with the repair process operating with MMS. The difference in the dose-effect relations of X-irradiation as compared with MMS may be brought back to the fact that X-rays, in contrast with MMS, produce double-strand breaks.

Factors affecting the induction of micronuclei at low doses of x-rays, mms and dimethylnitrosamine in mouse erythroblasts.

In erythrocytes from mouse bone marrow the time schedule of micronucleus formation in relation to the last DNA synthesis was investigate by [3H]thymidine labelling in the autoradiographic technique. The results suggest that micronuclei can be produced both in the G 2 and S periods by X-irradiation. Furthermore, X-rays had a delaying effect on the cell cycle leading to a pronounced under-estimation of the dose-effect curve at higher dosages. Even when the cells were harvested as late as 30 h after irradiation, the full effect had most likely not yet appeared at dosages over 100 rad. Combined treatment with caffeine did not influence the dose-effect curve of X-rays, indicating no influence of a caffeine-sensitive repair mechanism. The induction of micronuclei by MMS, in contrast with the effect of X-rays, seems to have been restricted, at least predominantly, to the period of DNA synthesis. The dose-effect relation of MMS was characterized by a threshold giving a weaker effect than expected at low doses. Pretreatment with caffeine enhanced the effect of MMS at high but not at low doses, suggesting an error-free repair process operating at low doses and an error-prone and caffeine-sensitive repair at higher doses. The extent of alkylation in the bone-marrow cells was linear with respect to injected dose of MMS both in the presence and absence of caffeine. Pretreatment with phenobarbital reduced the effect of MMS sixfold, which can be explained by a reduction of alkylation found in the bone marrow. This result is in agreement with the enhanced excretion of MMS or its metabolites into urine and bile after pretreatment of the mice with phenobarbital. DMN had no measurable effect on the frequency of micronuclei. However, in the presence of caffeine a significant effect was observed, which was roughly of the same magnitude for the two dosages used. Pretreatment with phenobarbital also indicated a synergistic effect between DMN and phenobarbital. The treatment with DMN, phenobarbital and caffeine together gave a frequency of micronuclei not different from the control level, suggesting some antagonistic action between phenobarbital and caffeine. The indication that DMN is caffeine-sensitive at low dosages, whereas MMS is not, might be related to the difference in the alkylating properties of these chemicals.

Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells.

A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two different laboratories, 17 and 59 mutants in sets 1 and 2, respectively, under different cell culture conditions. Mutation analysis was performed by amplification of hprt cDNA with the polymerase chain reaction and direct sequencing of the products. The data obtained showed similar spectra of spontaneous mutations in both sets of mutants, suggesting that culture does not play a major role in spontaneous mutagenesis. The majority of the mutations were base substitutions (greater than 60%), with twice as many transversions as transitions. Base changes were evenly distributed throughout the structural gene, including the splice junctions. All types of base substitutions appeared in comparable frequencies, except for A.T to T.A transversions, which were almost absent. The fraction of deletion mutations was low (13%). A striking feature of the observed mutation spectra is that one third of the spontaneous mutations analyzed involved aberrant splicing of the hprt primary transcript, with exon 4 being affected most frequently, indicating that splice mutations are a common mechanism of mutation in the hprt gene.

Inhibition of DNA synthesis is a potent mechanism by which cytostatic drugs induce homologous recombination in mammalian cells.

Recombination is a process thought to be underlying genomic instability involved in carcinogenesis. This report examines the potential of cytostatic drugs to induce intrachromosomal homologous recombination. In order to address this question, the hprt gene of a well-characterized mammalian cell line was employed as a unique endogenous marker for homologous recombination. Commonly used cytostatic drugs with different mode of action were investigated in this context, i.e. bifunctional alkylating agents, inhibitors of DNA synthesis, inhibitors of topoisomerases and a spindle poison. With the exception of the spindle poison, all these drugs were found to induce homologous recombination, with clear differences in their recombination potency, which could be related to their mechanism of action. Bifunctional alkylating agents were the least efficient, whereas inhibitors of DNA synthesis were found to be the most potent inducers of homologous recombination. This raises the question whether these later drugs should be considered for adverse effects in cancer chemotheraphy.

Mutagenicity testing on Chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermanns system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

Arsenic[III] and heavy metal ions induce intrachromosomal homologous recombination in the Hprt gene of V79 Chinese hamster cells

In the present study the carcinogenic metal ions Cd[II], Co[II], Cr[VI], Ni[II], and Pb[II], as well as As[III], were examined for their ability to induce intrachromosomal homologous and nonhomologous recombination in the hprt gene of two V79 Chinese hamster cell lines, SPD8 and Sp5, respectively. With the exception of Pb[II], all of these ions enhanced homologous recombination, the order of potency being Cr>Cd>As>Co>Ni. In contrast, Cr[VI] was the only ion to enhance recombination of the nonhomologous type. In order to obtain additional information on the mechanism of recombination in the SPD8 cell line, individual clones exhibiting metal‐induced recombination were isolated, and the sequence of their hprt gene determined. These findings confirmed that all recombinogenic events in this cell line were of the homologous type, involving predominantly a chromatid exchange mechanism. The mechanisms underlying the recombination induced by these ions are discussed in relationship to their genotoxicity, as well as to DNA repair and replication. Induced recombination may constitute a novel mechanism for induction of neoplastic disease. Environ. Mol. Mutagen. 35:114‐122, 2000

Relationship between chemical damage of DNA and mutations in mammalian cells: I. Dose-response curves for the induction of 6-thioguanine-resistant mutants by low doses of monofunctional alkylating agents, x-rays and UV radiation in V79 Chinese hamster cells

The shape of the dose-response curve for mutations induced at low doses of mutagenic agents in mammalian cells was studied. With the exception of X-rays and MMS, which are very toxic in relation to their mutagenic potency, dose-response studies with EMS, MNU, ENU and UV radiation were performed at doses giving about 100% survival. The results from several experiments were pooled for each agent to get higher resolution power at low doses, and the result was compared with what could be expected when linear interpolation is performed from higher doses. The dose response for induction of mutations by UV- and X-irradiation did not deviate from linearity at low doses. In contrast with irradiation and the ethylating agents EMS and ENU, the methylating agents MMS and MNU showed a significantly lower effect at low doses as compared with estimation from a dose 5 times as high. The extent of alkylation of DNA by various doses of MMS was linear in the same dose intervals. It is suggested that the decreased response at low doses of the methylating agents found here may be connected with changes in the mechanism of repair of the lesions induced at different dose levels.