Spectrum ofcis-diamminedichloroplatinum(II)-induced mutations in a shuttle vector propagated in human cells

Abstract

The supF gene of the shuttle vector pZ189 was used as a target for the study of mutations induced by cis‐diamminedichloroplatinum(II) (cis‐DDP). Normal human repair‐proficient fibroblasts and cis‐DDP repair‐deficient xeroderma pigmentosum (XP) cells were used as host cells to study the effect of cis‐DDP on the inhibition of shuttle vector replication and mutagenesis. Transfection of cis‐DDP‐treated pZ189 into normal and XP cell lines resulted in a marked increase in the mutation frequency and a decrease in the replication efficiency of the vector. However, these effects were much greater for the plasmid propagated in XP cells. Atomic absorption spectroscopy showed that six to eight Pt‐DNA adducts per plasmid were necessary to inhibit plasmid replication by 50% in normal cells. In contrast, only one to two Pt‐DNA adducts were necessary to inhibit replication of the plasmid by 50% in XP cells. Analysis of mutation sites demonstrated that cis‐DDP treatment resulted primarily in single and double mutations separated by one base and limited to a few locations within the 85‐bp mature tRNA. Propagation of the cis‐DDP‐treated vector in either normal or XP cells led to predominantly transversion mutations at AGA, AGG, and GAG sites and a cis‐DDP‐associated deletion of 174 bp. Although mutations occurred at target sites for cis‐DDP adduct formation, there was no correlation between sites of mutation and the most frequent sites of adduct formation.