Mutation Research, 241 (1990) 369-377 Elsevier

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MUTGEN 01573

Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-l-nitrosourea in Saccharomyces cerevisiae L y n n e t t e R. F e r g u s o n Cancer Research Laboratory, University of Auckland Medical School, Private Bag. Auckland (New Zealand) (Received 24 November 1989) (Revision received 6 March 1990) (Accepted 12 March 1990)

Keywords: DNA repair, inhibition; 1,3-Bis(2-chloroethyl)-l-nitrosourea; Saccharornyces cerevisiae

Summary The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-l-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential. Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion. BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain. However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential. Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative. Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy. Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU. Hydroxyurea actually decreased toxicity of the BCNU. Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug. Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.

It is well recognised that lesions induced by alkylating agents are subject to DNA repair. Many alkylating agents play an important role in cancer

Correspondence: Dr. Lynnette R. Ferguson, Cancer Research Laboratory, University of Auckland Medical School, Private Bag, Auckland (New Zealand).

chemotherapy, and it must be considered that repair of the lesions induced will decrease the toxicity and therefore the therapeutic effectiveness of the drugs. Additionally, DNA-repair processes appear to be involved in undesirable manifestations of drug action such as mutagenicity and mitotic crossing-over. Alkylating agents used in the clinic are known to be potent carcinogens and this is likely to relate to their mutagenic and

0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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recombinogenic potential (Schmahl et al., 1986; Kaldor et al., 1988; Ferguson et al., 1989). The possibility has been raised that the use of DNA-repair inhibitors might prevent the repair of lesions induced and thereby lead to maximum efficiency (in terms of increased toxicity) of chemotherapeutic anticancer drugs (e.g. Grigg et al., 1977; Kann et al., 1980; Roberts, 1984). Most alkylating agents in the clinic are bisalkylating drugs, with the ability to cross-link D N A (Connors, 1986). It has been previously found that cross-linking agents have an increased ability to cause mitotic crossing-over as compared to related monoalkylating drugs (Averbeck and Moustacchi, 1979; Ferguson and Turner, 1988). There is good reason to believe that mitotic crossing-over is coordinated independently of other mutagenic events (Fabre and Roman, 1977; Fahrig, 1979), and it has been revealed as an important mechanism for activation of antioncogenes (Cavenee et al., 1986). There is currently only limited information available as to whether DNA-repair inhibition will reduce either mutagenic potential or ability for mitotic crossing-over in cross-linking drugs which are used as clinical antitumour drugs. In this study, we have used S. cerevisiae strain D5 as a model system, in order to test whether DNA-repair inhibitors can not only increase toxicity but also reduce mutagenic potential and ability for mitotic crossing-over of a bisalkylating drug. BCNU has been selected as a commonly used clinical agent with D N A cross-linking ability (Kohn, 1981). It should be noted, however, that Kunz and Mis (1989) have evidence that monoadducts play a significant role in the mutagenesis by this drug.

Saccharomyces cerevisiae The diploid strain D5 (Zimmermann, 1973) was kindly provided by Dr. B.S. Cox (Botany School, University of Oxford). This strain has the genotype: a ade2-40 a ade2-119

D5:

The related BK series were very kindly constructed by and provided to us by Dr. B.A. Kunz, University of Manitoba, Canada (BK0, BK3 and BK18) and Dr. Darlene Ager, Dept. of Biology, York University, Toronto (BK52). Characteristics and methods for the construction of BK0, BK3 and BK18 have been previously published by Kunz and Haynes (1982). The rad52 strain was derived as follows: The rad52 replacement plasmid (pSM22) was obtained from David Schild at the University of California at Berkeley. Ura3-52 was crossed into the haploid strains used to make BK0 and then the RAD52 gene in each of the resulting haploids was replaced with the URA3 disruption of RAD52. Disruption of the rad52 gene was confirmed by sensitivity to methyl methanesulfonate and by D N A hybridisation analysis. The rad52 haploids were then crossed to each other to make BK52. The four strains had the following genotypes: BK0:

a ade2-40 a ade2-119

cyh2 trp5-12 ilvl-92 CYH2 trp5-27 ilvl-92

BK3:

a ade2-40 a ade2-119

cyh2 trp5-12 ilvl-92 rad3-2 CYH2 trp5-27 ilvl-92 rad3-2

BK18:

a ade2-40 a ade2-119

cyh2 trp5-12 ilvl-92 rad18-2 CYH2 trp5-27 ilvl-92 rad18-2

a

cyh2 trp5-12 ilol-92 ura3-52 rad52:URA3 CYH2 trp5-27 ilvl-92 ura3-52 rad52:URA3

B K 5 2 : -a

ade2-40 ade2-119

Materials and methods

Drugs BCNU was purchased from Bristol Laboratories, New York (U.S.A.), cycloheximide from Boehringer, Mannheim (West Germany), caffeine from Sigma Chemical Co., Missouri (U.S.A.), hydroxyurea from Squibb, New Jersey (U.S.A.). Amsacrine was kindly synthesised in this laboratory by Dr. G.J. Atwell.

For each of these strains, a single colony isolate was inoculated into liquid yeast complete medium (YC; Cox and Bevan, 1962) and grown to stationary phase for 24 h. Dimethyl sulphoxide was added to 10%, 1-ml aliquots were frozen to - 7 0 ° C , and stored at this temperature before use. For all experiments, the 1-ml sample was thawed, added to 10 ml of fresh medium and grown for exactly 2 h before use.

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Microtitre assay for mutation and mitotic crossingover This assay has been described in detail by Ferguson (1984). Briefly, a log-phase culture was washed, then diluted into 0.87% saline. A 96-well microtitre tray ( A / S Nunc, Denmark) was inoculated with (usually) 100-/~1 aliquots of the diluted yeast culture, and drugs added at various dilutions to the wells, to a m a x i m u m of 1000 # g / m l . Drugs were dissolved in dimethyl sulphoxide, and dilutions made so that there was no more than 1% D M S O in each well. Trays were incubated for the indicated time at 3 0 ° C , an appropriate dilution made from each well into saline, and 100/~1 plated onto each of 10 YC plates. Cell numbers were calculated so that the dilutions at this point were at least 1/104 , thereby effectively washing drugs from cells by dilution (Ferguson, 1984). Plates were incubated at 30 ° C for 5 days, and scored for ordinary, coloured or sectored colonies by visual i n s p e c t i o n ( Z i m m e r m a n n , 1973). C o l o n i e s attributable to mitotic crossing-over are r e d / p i n k or r e d / p i n k / w h i t e , while all other coloured colonies result mainly from mutation but also from gene conversion and other events. These events

are included in the score for 'total aberrant' colonies. All experiments were performed at least twice and the data compared for reproducibility. The presented data have been pooled. Each experimental point presented for aberrations is based on an average count of 10000 colonies per data point, and never less than 1000 colonies.

Inhibitor experiments It had originally been intended to treat with BCNU, followed by treatment with the inhibitor in question. However, because a minimum of 2 h drug treatment was found essential to reveal genotoxic effects and a considerable amount of D N A repair could occur during this time, inhibitors were included directly with B C N U in these experiments. Otherwise, treatment and plating conditions were as described above. Results

Characteristics of toxicity, mitotic crossing-over and other events by B C N U in strain D5 In initial experiments, yeast cells were incubated with a single concentration of (233 /~M)

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Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2...
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