Repair of O⁶-Methylguanine in Rat Pancreatic β-Cells After Exposure to N-Methyl-N-Nitrosourea

Because of the possible involvement of O⁶-methyldeoxyguanosine as a cytotoxic and carcinogenic lesion in pancreatic β-cells, studies were undertaken to assess the ability of rat β-cells to repair this DNA lesion. Primary cultures of neonatal rat β-cells were shown to contain very low levels of O⁶-methylguanine-DNA-methyltransferase activity, the predominant mechanism for repairing O⁶-methyldeoxyguanosine in mammalian cells. However, using a ³²P-endlabeling assay to measure O⁶-methyldeoxyguanosine in cells after exposure to N-methyl-N-nitrosourea, it was determined that rat β-cells repaired O⁶-methyldeoxyguanosine to a substantial extent over a 24-h period. To elucidate the mechanism of O⁶-methyldeoxyguanosine repair in the virtual absence of constitutive O⁶-methylguanine-DNA-methyltransferase expression, studies were performed to determine if O⁶-methylguanine-DNA-methyltransferase expression was enhanced in N-methyl-N-nitrosourea-treated β-cells. No increase in O⁶-methylguanine-DNA-methyltransferase activity was detected 24 or 48 h after exposure. However, Northern blot analysis showed a two- to threefold elevation in O⁶-methylguanine-DNA-methyltransferase messenger RNA levels in β-cells 12 and 24 h after N-methyl-N-nitrosourea treatment. This finding is the first demonstration of a change in O⁶-methylguanine-DNA-methyltransferase messenger RNA levels in a cell type with low constitutive activity.