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Overexpression of Metallothionein in Chinese Hamster Ovary Cells and Its Effect on Nitrogen Mustard-induced Cytotoxicity: Role of Gene-specific Damage and Repair

Karsten Wassermann¹, Miroslav Pirel² and Vilhelm A. Bohr³

Laboratory of Molecular Pharmacology, Developmental Therapeutic Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892

³ Present address: Laboratory of Molecular Genetics, National Institutes on Aging, NIH, 4940 Eastern Ave., Baltimore, MD 21224. To whom requests for reprints should be addressed.

Overexpression of metallothionein in mammalian cells has been associated with protection from cytotoxic chemicals and acquired resistance of tumors to cytotoxic drugs. The mechanism of this effect, however, remains unclear. We have explored whether cytotoxicity of the bifunctional alkylating agent nitrogen mustard was correlated with the extent of DNA damage formation and repair in the metallothionein gene regions in Chinese hamster ovary cells. The DNA damage and repair were examined in metallothionein-overexpressing, cadmium-resistant Chinese hamster ovary cells, Cd^r200T1, with or without zinc-induced transcriptional activation, and in the parental CHO-met- cell line. The zinc-induced Cd^r200T1 cells tolerated significantly higher doses of nitrogen mustard than did the uninduced Cd^r200T1 variant. The parental CHO-met- cells, which did not have any detectable metallothionein expression, were even more resistant to nitrogen mustard than the zinc-induced Cd^r variants. Nitrogen mustard-induced N-alkylpurines were formed with a higher frequency in inactive genomic regions than in the active genes. The removal of N-alkylpurines was similar in the active MT I gene region in Cd^r200T1 and the silent MT I gene region in the parental cells, and the expression of these genes was determined by Northern assay. The MT II gene-containing region was repaired less efficiently than the MT I gene, independently of zinc induction. Further, preferential repair of nitrogen mustard-induced N-alkylpurines were detected in a single copy of the essential active dihydrofolate reductase gene as compared to a downstream noncoding region. This preferential repair was unaffected by the presence of zinc. Neither damage formation nor repair kinetics in the MT gene regions seemed to parallel the observed spectrum of sensitivity to HN2.

¹ Present address: Department of Biology and Toxicology, Danish National Institute of Occupational Health, Lersø Parkallé 105, DK-2100 Copenhagen Ø, Denmark. Supported by fellowship grants from the Danish Medical Research Council (12-9352) and the Danish Cancer Society (89-089, 92-030).

² On leave from Cancer Research Institute, Slovak Academy of Sciences, Department of Molecular Genetics, pitálska 21, 81232 Bratislava, Czechoslovakia. Supported by an American Cancer Society Eleanor Roosevelt Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/16/92. Accepted 10/ 6/92.

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