Mutagenesis of H-ras codons 11 and 12 in human fibroblasts by N-ethyl-N-nitrosourea.

Abstract

Codon 12 of the ras protooncogenes represents a mutational hotspot in human cancer. A distinct pattern of base pair changes has been observed that may allow insight into the class of carcinogens that induced the mutations. In an attempt to identify candidate carcinogens we have studied the mutability of codons 11 (GCC) and 12 (GGC) of the c-H-ras1 gene in human foreskin fibroblasts to N-ethyl-N-nitrosourea (ENU) by restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR). This genotypic mutation system allows the quantitation of low frequencies of mutated sequences without expansion and phenotypic selection of mutated cells. ENU induced the transition of G to A in the middle position of codon 12 with highest efficiency. This is the same mutation that is found predominantly in human tumors. These mutations are most likely the consequence of miscoding opposite O6-ethylguanine adducts and our data indicate strong bias for the coding strand. No evidence was obtained for spontaneous deamination of the CpG-dinucleotide despite its high content in 5-methylcytosine.