Activated c-Ha-ras protooncogenes have recently been identified in the
DNA of some spontaneous hepatic
tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in
DNA from well-differentiated
hepatomas initiated by a single dose of
carcinogen given to male B6C3 F1 mice at 12 days of age.
DNA from each of 25
hepatomas, induced by N-hydroxy-2-acetylaminofluorene,
vinyl carbamate, or
1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by
DNA from 24 of these
hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other
tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of
p21 ras proteins in NIH 3T3 transformants derived from a majority of the
hepatomas suggested that the activating mutations were localized in the 61st
codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of
codon 61 was detected in
DNA from primary
tumors and NIH 3T3 cells transformed by
DNA from 6 of 7
vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced
hepatomas. Selective
oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st
codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced
hepatomas. By the same criterion, an AT----GC transition at the second position of
codon 61 was the activating mutation in 1 of 7
vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced
tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical
carcinogens with this gene in vivo.