Activation of N-ras induced by ultraviolet irradiation in vitro

Abstract

Ultraviolet (UV) light is an important environmental mutagen and one of the major factors in the etiology of skin cancer in man. In this study we have addressed the question whether UV light can activate in vitro the N-ras proto-oncogene into an active oncogene, and whether pyrimidine cyclobutane dimers or (6-4) photoproducts are the premutagenic lesion. To that end, the cloned human N-ras proto-oncogene (pN-ras) was irradiated with UV light (254 nm) which results in the formation of cyclobutane dimers and (6-4) photoproducts and the irradiated DNA was transfected into Rat-2 cells. Whereas untreated pN-ras never gave foci upon transfection into Rat-2 cells, UV-irradiated pN-ras (100-300 J.m-2) yielded 1-2 foci/micrograms DNA. The DNAs of 49 primary foci were subjected to a second round of transfection. 13 of these were positive. The DNAs that did not give rise to secondary foci invariably contained high copy numbers of pN-ras. The DNAs from the secondary foci had only a low copy number of pN-ras. Using synthetic oligonucleotide probes we have found that all N-ras genes that gave rise to secondary foci contained a mutation at positions in codon 12 or 61. Only one primary transformant, negative in the secondary transformation assay, was shown to harbor a mutated N-ras gene. From these results we conclude that UV light can activate N-ras genes. Mutations were preferentially found at the 61st codon (11), harboring a TT doublet, while only 3 mutations were found at the 12th codon and none at the 13th codon, both harboring CC doublets. Of the 14 point mutations analysed 6 represented transitions and 8 transversions. No specific base-substitution could be regarded as predominant. Furthermore, treatment of the irradiated N-ras plasmids with photoreactivating enzyme prior to transfection, which specifically monomerizes cyclobutane dimers but not other photoproducts, reduced the transformation frequency several fold. None of the 36 DNAs from the primary foci obtained after transfection of photoreactivating enzyme-treated N-ras gave rise to secondary foci. These DNAs all contained high copy numbers of unmutated pN-ras. From this result we conclude that cyclobutane dimers, and not (6-4) photoproducts, are the major premutagenic lesions, responsible for the activation of N-ras. The possible role of ras genes as targets for UV-induced carcinogenesis is discussed.