Amplification of the DNA repair gene O6-methylguanine-DNA methyltransferase associated with resistance to alkylating drugs in a mammalian cell line.

The cytotoxic action of such alkylating chemotherapeutic drugs as 2-chloroethyl-N-nitrosourea (CNU) derivatives is countered by the repair protein O6-methylguanine-DNA methyltransferase (MGMT), which removes O6-alkylguanine induced in the DNA by these agents. Resistance to these drugs is often correlated with the MGMT levels in normal and tumor cells of human and rodent origin. Exposure of mouse 3T3 cells to increasing concentrations of CNU, and subsequent selection of resistant cells, led to the isolation of clones with 5-10 times higher levels of MGMT activity than in the control. The increased MGMT expression at both mRNA and protein levels resulted from 5- to 10-fold amplification of the Mgmt gene. Amplification of this gene was not associated with concomitant amplification of another alkylation damage repair gene, N-methylpurine-DNA glycosylase. No amplification of at least three other genes on chromosome 7 (which contains the Mgmt gene) was observed in the drug-resistant cells. Furthermore, the amplified Mgmt sequence was not associated with a homogeneously staining region, or double minute chromosomes, nor present as episomal DNA. In situ hybridization of metaphase chromosomes of the drug-resistant cells indicated both translocation and localized amplification of the Mgmt gene.