Quantification of O6-methylguanine-DNA methyltransferase mRNA in human brain tumors

Abstract

O6-Methylguanine-DNA methyltransferase (MGMT) is strongly involved in drug resistance mechanism of tumor cells to chloroethylnitrosoureas (CENUs), because it removes and repairs CENU-induced O6-alkylguanine-DNA by accepting the alkyl group at a cysteine moiety. MGMT activity and MGMT mRNA expression are good indicators for detection of sensitive cells or resistant cells to CENUs. In the present study, we applied a non-radioactive reverse transcription-polymerase chain reaction (RT-PCR) method on quantitative measurement of MGMT mRNA expression. Estimated levels of MGMT mRNA expression determined by this RT-PCR method were consistent with the actual doses of MGMT mRNA. This relationship was noted at a wide range from 10 fg to 10 pg. The relative expression levels of MGMT mRNA estimated from kinetic analysis correlated well with MGMT activity determined using 3H-methyl-nitrosourea-treated DNA substrate in brain tumor cells (P < 0.001 with a correlation coefficient of 0.997). The RT-PCR method facilitated quantitative measurements in even a small amount of biopsy specimens obtained by stereotactic brain surgery.