COMPARISON OF O(6)-METHYLGUANINE-DNA METHYLTRANSFERASE MESSENGER-RNA LEVELS IN HA-RAS MUTATED AND NON-MUTATED RAT MAMMARY-TUMORS INDUCED BYN-METHYL-N-NITROSOUREA

Activation of the Ha-ras oncogene in N-methyl-N-nitrosourea (MNU))-ind uced rat mammary tumors has been well documented. Such Ha-ras activati on is thought to be brought about by direct action of carcinogens resu lting in a G-->A transition at the second nucleotide of codon 12. Howe ver, a DNA repair enzyme, O-6-methylguanine-DNA methyltransferase (MGM T), can specifically remove methyl groups from O-6-methylguanine, whic h is a major mutagenic and carcinogenic DNA lesion leading to the G--> A transition. In this study, we compared the amount of MGMT mRNA in MN U-induced rat mammary tumors with and without such Ha-ras activation. A single injection of MNU into 82 female Sprague-Dawley rats induced 8 0 mammary carcinomas. RNase protection analysis and subsequent sequenc ing revealed that 42 of 65 randomly selected tumors contained Ha-ras o ncogenes activated by the G-->A transition. The amount of MGMT mRNA wa s then measured by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) amplification and Southern hybridization. No o bvious difference in the level of MGMT mRNA was detected between the t wo tumor groups. In addition, in the course of our experiment, five of 42 tumors classified as containing activated Ha-ras oncogenes proved to contain low percentages of tumor cells with the Ha-ras activation. These results suggest that Haras activation in MNU-induced rat mammary tumors may not necessarily be influenced by differences in MGMT activ ity. They also raise the possibility that activation of other oncogene s and/or inactivation of unidentified tumor suppressor gene(s) may be involved in development of a certain proportion of tumors with activat ed Ha-ras oncogenes, as is suspected in the case of tumors without Ha- ras activation.