Mutational activation of H- ras oncogene transformability by alkylnitrosourea-induced DNA damage

Abstract

To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H- ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1–5 mM) and ENU (1–15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H- ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/μg MNU alkylated and 1.6 to 0.6/μg ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/ MspI restriction length polymorphism (RFLP) at codon 12 of H- ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H- ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H- ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.