In vitro mutational specificity of cisplatin in the human hypoxanthine guanine phosphoribosyltransferase gene.

The in vitro mutational spectra of cisplatin [cis-diamminedichloroplatinum(II)] in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lymphoblasts was examined by a combination of polymerase chain reaction and denaturing gradient gel electrophoresis. Several thousand independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase locus by cisplatin and were selected en masse by addition of 6-thioguanine to the bulk culture. Polymerase chain reaction was used to amplify exon 3 from the complex mutant population, and denaturing gradient gel electrophoresis was used to separate wild-type DNA sequences from mutant sequences. Mutational hotspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the complex population represented by the novel bands. The mutant bands were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of several thousand mutants were determined. Cisplatin produced several mutational hotspots in exon 3. About 9-10% of the cisplatin-induced mutants had mutations in a GGGGGG sequence (base pairs 207-212). GC----AT substitutions at the second and third guanines in the 5'-GGGGGG-3' run made up about 2 and 4% of the induced mutants, respectively. About 4% of the induced mutants contained a GC----TA substitution at the sixth guanine. About 1% of the cisplatin-induced mutants had an AT----TA transversion in a TAGA sequence (base pair 271; mutated base is underlined). Our results are consistent with mutations occurring at GpG and ApG sites. These nucleotide sequences have been identified as the primary sites of cisplatin adduction.