IN-VITRO ACTIVATION OF THE HUMAN HARVEY-RAS PROTOONCOGENE BY AFLATOXIN B-1

Activation of ras proto-oncogenes occurs frequently in vivo in chemica lly induced rodent tumours, including rat hepatomas induced by aflatox in B-1. This study examines the in vitro activation of a human ms gene by this mycotoxin, A plasmid containing the human Ha-ms proto-oncogen e, together with a neomycin resistance gene (pECneo), was incubated in vitro with a microsomal system generating aflatoxin B-1 8,9-epoxide, Subsequent transfection of the plasmid into mouse NIH 3T3 fibroblasts, followed by G418 selection and s.c. injection of surviving cells into immunodeficient mice demonstrated that the proto-oncogene had acquire d transforming capacity, Although a single tumour resulted from simila r treatment of incubated unconjugated plasmid, no tumours were produce d by a secondary round of transfections using DNA from this tumour, Se lective PCR amplification of the human Ha-ras gene in extracted tumour DNA followed by sequencing demonstrated the presence of G-->T transve rsions either at the first or middle base of codon 12 in tumours resul ting from transfection with the aflatoxin-B-1-modified pECneo plasmid, but this was not detected in the single tumour resulting from transfe ction with the unmodified plasmid, Thus, although a mutation in the Ha -ras gene has not been reported for human primary hepatomas occurring in aflatoxin-exposed populations, metabolically activated aflatoxin B- 1 is capable of mutating this proto-oncogene to its oncogenic form in vitro, No mutations were observed in codon 61. It appears that, in con trast to the frequently reported G-->T transversions in codon 249 of t he p53 gene in primary hepatomas in aflatoxin-exposed humans, the fail ure to detect Ha-ms mutations in these tumours is not due to an inabil ity of aflatoxin B-1 to activate this protooncogene, The G-->T transve rsions observed in this study contrast with the most frequent aflatoxi n B-1 in vivo induced mutations, G-->A transitions in the rat Ki-ras g ene, Possible mechanisms for these differences are discussed.