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 p21ras protein.

Mammalian assay for site-specific DNA damage processing using the human H-rasproto-oncogene

“…Also, the results of this experiment strongly suggest a low copy number of plasmid per cell: the 2-fold increase in foci at 99:1 wt to activated H-ras ratio did not derive from multiple copies of wt H-ras expression (37), as the same concentration of wt H-ras alone does not produce foci above background of non-transfected NIH 3T3 cells ( The highest correction rate of G:T-+G:C (96.4%) agrees with previous findings by other investigators who have described more than one error correction mechanism for this mismatch, resulting in more accurate repair than in other types of mismatches (7)(8)(9). The lowest correction rate of T:C-+G:C (67%) agrees with human ras mutation literature which cites codon 12; middle G:C -+T:A transversion mutations as the most frequent (21,25,40). The majority of all mixed results appear to be a 1:1 ratio of each base pair by sequencing results.…”

“…It is interesting that the T:C mismatch is corrected to G:C with only 67% efficiency. Considering that human ras mutations at codon 12 or 13 frequently undergo G:C-+T:A transversions (21,25,40), we could speculate that tumors containing this mutation have a mutator phenotype specifically involving T:C mismatches at this location. We found mixtures of both wild-type and mutated codon 12 sequences in 3.6% ofG:T mismatch samples, 10o of A:C mismatch samples and 15% of T:C mismatch samples.…”

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

“…To address this question, we have investigated the ability of purified calf thymus DNA polymerases a, (3, 8, and s to catalyze in vitro the bypass synthesis of a single Pt-d(GpG) adduct placed on codon 13 of the human HRAS protooncogene, the same sequence used for our previous in vivo studies (10). Results show that only DNA polymerase 3 is capable of in vitro translesional synthesis and indicate that its ability to initiate DNA replication opposite the cisplatin adduct may be a key step in this process.…”

“…vector bearing a unique intrastrand bifunctional adduct Ptd(GpG) at codon 13 of the human protooncogene HRAS is replicated in simian COS-7 cells and that such translesion synthesis may be mutagenic (10).…”

“…To address this question, we have investigated the ability of purified calf thymus DNA polymerases a, (3,8, and s to catalyze in vitro the bypass synthesis of a single Pt-d(GpG) adduct placed on codon 13 of the human HRAS protooncogene, the same sequence used for our previous in vivo studies (10 (8-mer, 14-mer, 38-mer, and the complementary 52-mer) were annealed by mixing an excess of the 14-mer, 52-mer, and 5'-phosphorylated 38-mer with either the 5'-phosphorylated 8-mer or-the 5'-phosphorylated 8-mer-Pt adduct oligonucleotides, which had been purified and characterized as described (16). After ligation the resulting 60-mer or 60-mer-Pt substrates were purified on a 20% polyacrylamide/7 M urea/30% formamide denaturing gel.…”