Hypermethylation of CpG sites within the promoter region of the O6-methylguanine-DNA
methyltransferase (MGMT) gene occurs frequently in human
cancer, preventing both MGMT expression and repair of alkylation damage. To assess the role of MGMT in the development of mouse skin
tumors induced by initiation-promotion protocols, methylation of the MGMT promoter was examined in
tumor DNA using methylation-specific PCR. To determine whether MGMT promoter methylation was affected by the
tumor induction protocol,
tumors were initiated by
N-methyl-N'-nitro-N-nitrosoguanidine (
MNNG) or 7,12-dimethylbenz[a]
anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or
mezerein. Although the MGMT promoter was not methylated in normal skin, promoter methylation was found in 56 of 136
papillomas (41.2%) and in 19 of 37
squamous cell carcinomas (51.4%). When methylation of the MGMT promoter was compared in the 4 treatment groups, hypermethylation was found more frequently in
tumors initiated by DMBA and promoted by
mezerein, a protocol associated with a high frequency of malignant conversion. Methylation was found in some
tumors as early as 5 weeks after initiation, but the methylation frequency increased with time. MGMT promoter methylation reduced MGMT expression as determined by immunohistochemistry. Although MGMT promoter methylation was not generally correlated with ras mutations, the frequency of MGMT methylation was higher in
MNNG-initiated,
mezerein-promoted
papillomas with mutations in Ha-ras compared to
papillomas with Ki-ras. Methylation of the MGMT promoter, associated with reduced MGMT expression, is found in nearly half of mouse skin
tumors, but varies with both the
tumor initiator and
tumor promoter, and may be a key step in the progression from
papillomas to
carcinomas.