Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.

Abstract

We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including 06-methylguanine and 06-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TKcells. Our results indicate that both types of 06-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TKV colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both 0-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA -. GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair.

Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA methyltransferase in human cancer

“…Thus, O 6 -methylguanine is read as an adenine and mispairs with thymine (Horsfall et al, 1990). Supporting these data, the most common mutations caused by alkylating agents are G : C to A : T transitions (Horsfall et al, 1990), exemplified in the frequent generation of G to A transitions in the oncogene K-ras when the carcinogen N-methylnitrosourea (that forms O 6 -methylguanine adducts) is used in experimentally induced tumor systems (Sukumar et al, 1983;Mitra et al, 1989). Avoidance of the mutagenic effect is directly related to the presence of a functional MGMT protein (Pegg et al, 1995).…”

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

“…All mutations were found at the 2nd position of the 12th codon (rather than on the 1st position, see Figure 4). This latter ®nding also holds for ras gene mutations at the same codon in for instance MNNG-or MNU-induced mouse skin tumors (Mitra et al, 1989;Brown et al, 1990Brown et al, , 1995. Further analysis of the same set of 28 tumors revealed that additionally one papilloma and one SCC carried a K12-ras gene mutation (also a G?A transition at the 2nd position of the codon, results not shown).…”

“…Alkylating agents, for example, when applied on the skin of wild type mice, induce papillomas by targeting the 2nd G residue in codon 12 of the H-ras gene, resulting in a G?A transition like we found in U.V.B.-induced papillomas of XPA-de®cient mice ( Figure 5 and references. Mitra et al, 1989;Brown et al, 1990Brown et al, , 1995. Furthermore, the main genetic target of the classical skin carcinogen DMBA in papilloma development, is an A-residue in codon 61 of the mouse Hras gene (Brown et al, 1995).…”

Stimulated human leukocytes cause activating mutations in the K-ras proto-oncogene

“…To assess the ability of 8 hydroxydeoxyguanosine to induce K-ras oncogene activation, we utilized the pGM 22 mammalian expression vector (Mitra et al, 1989), which contains codons 1 ± 8 and codons 14 ± 189 of a nontransforming (codon 59=Ala) v-H-ras clone, separated by a 1.3 kb spacer fragment. pGM 22 was digested with BspMI to remove the spacer fragment, and a full-length ras gene, containing 8 hydroxydeoxyguanosine at codon 12, was reconstituted by ligating a synthetic double-stranded oligonucleotide (encoding codons 9 ± 13 of K-ras and containing 8 hydroxydeoxyguanosine substituted for deoxyguanosine at either the ®rst or second position of codon 12) into the BspMl site of PGM 22.…”