THE ROLE OF ACTIVE AND ACTIVE-SITE ALKYLATED HUMAN O6-METHYLGUANINE-DNA METHYTRANSFERASE (MGMT AND R-MGMT) IN DNA REPAIR AND BIOMONITORING

Abstract

Monoclonal and polychlonal antibodies raised against recombinant human MGMT were characterized and used to study the role of active and active-site alkylated human O6-methylguanine-DNA methyltransferase (MGMT and R-MGMT) in DNA repair and biomonitoring. The human DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT, Mr 22kd), which protects cells against the mutagenic effect of alkylating carcinogens, was found to be localised in the nucleus (except the nucleolus) by immunofluorescence staining using polyclonal and monoclonal antibodies. The supporting experiments came from differential staining of the GMT-deficient (mer-) and proficient (mer+) cells, Western blotting analysis, and specific antibody depletion studies with the immobilised fusion protein, GSTMGMT-glutathione-Sepharose. Its localisation in the nucleus agrees with its biological function and possibly explains the ineffective protection of mammalian cells (mer-) transfected with the E. coli MGMT gnes from bifunctional alkylating agents. As MGMT-deficient tumour and normal tissue are known to exist, the availability of these antibodies would serve as a useful prognostic tool for determining the success of choloroethylnitrosourea (CENU) chemotherapy. Cells resist the major mutagenic effects of alkylating agents by the action of O6-methylguanine-DNA methyltransferase (MGMT), which transfer the alkyl (R) group of O6-alkylguanine, produced in DNA by these chemicals, to a cysteine residue in its active site (formation of R_MGMT). cellular R_MGMT (which represents a record or memory within the cells exposed to these chemicals) as shown here, can be assayed by its sensitivity toward proteolysis by V8 protease. The possible use of this assay for monitoring exposure to alkylating carcinogens was investigated by using cultured cells and a preliminary study with the use of human blood from normal subjects and patients undergoing chemotherapy. Cultured cell experiments show that R-MGMT is sufficiently stable for monitoring purposes and its level bears a dose-response relationship to the concentrations of the alkylating agent used. Interestingly, experiments with blood from patients undergoing chemotherapy show a gradual formation of R-MGMT in cyclophosphamide treated patients. This methodology, which allows for the possible quantification of active MGMT (cellular DNA repair capacity) and R-MGMT ((recent exposure) simultaneously, is a potentially useful technique for monitoring exposure to alkylating carcinogens.