Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 700C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C G), 5 at the second (A T), and 5 at the third (G C). The point mutations induced by anti-BPDE were predominantly G C → T A and A T → T A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G C → T A, and a single depurination-induced activation that was analyzed contained an A T → T A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells