Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage.
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/microgram MNU alkylated and 1.6 to 0.6/microgram 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.
Yamasaki
E F
EF
Department of Radiology, Ohio State University, Columbus 43210.
Salamon
D P
DP
Wani
A A
AA
D016475:3T3 Cells; D000478:Alkylation; D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D005038:Ethylnitrosourea / Q000633:toxicity*; D005786:Gene Expression Regulation; D011905:Genes, ras; D008770:Methylnitrosourea / Q000633:toxicity*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation
D008770:Methylnitrosourea; D004247:DNA; D005038:Ethylnitrosourea