Mutagenesis in monkey cells of a vector containing a single d(GPG)cis-diamminedichloroplatinum(ll) adduct placed on codon 13 of the humanH-rasproto-oncogen

Abstract

Cisplatin (cis-{Pt(NH₃)₂Cl₂}) is a widely used antitumor agent whose mutagenic activity raises the possibility of the induction of secondary cancer as a result of treatment. Mutation of the proto-oncogene H-ras is found in more than 30% of all human tumors, where it has been postulated to contribute to the initiation and progression of human cancers. Activating mutations in the H-ras gene are predominantly single-base substitutions, most frequently at codons 12,13 and 61. In the present work we have studied the mutational spectra induced by a single cis-{Pt(NH₃)₂d(GpG)} adduct, the most frequent DNA crosslink formed by cisplatin. We have constructed a 25-mer-Pt oligonucleotide singly modified at codon 13 (GGT) within the human H-ras DNA sequence and we have inserted it into a single-stranded SV40-based shuttle vector able to replicate in simian COS7 cells. After replication in the mammalian host, vectors were extracted, amplified in bacteria and DNA from 124 randomly chosen colonies was sequenced. The observed mutation frequency was 21%. Base substitutions were the most frequent modification. 92% of the mutagenic events occurred at one or both of the platinated guanines of codon 13. The single G—T transversion accounted for 65% of the total mutations scored. All single base substitutions were located at the G in the 3′ position showing, for the first time, that the guanine at the 3′ side of a cis-{Pt(NH₃)₂d(GpG)} adduct may be a preferential site for cisplatin induced mutations. The substitution G—T at this position of the codon 13 of the H-ras proto-oncogene is known to induce the oncogenic properties of the p21^ras protein.