INTRACELLULAR-LOCALIZATION AND INTERCELLULAR HETEROGENEITY OF THE HUMAN DNA-REPAIR PROTEIN O-6 METHYLGUANINE-DNA METHYLTRANSFERASE

O-6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor res istance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunof luorescence microscopy, and fluorescent signals were quantified by dig ital image analysis. MGMT was found both in the cytoplasm and the nucl eus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT leve ls, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O-6-Benzylguan ine, a suicide substrate inactivator for MGMT activity, reduced MGMT i n human cells and in a mouse xenograft to levels undetectable by antib ody assay 1 h post-treatment. In melanoma specimens taken from a patie nt 3 h post-treatment with temozolomide, MGMT levels were reduced by 7 0%. This quantitative immunofluorescence assay can be used to monitor MGMT and its depletion in human tumors to improve the use of alkylatin g agents in cancer chemotherapy.