HETEROGENEITY OF O-6-ALKYLGUANINE-DNA-ALKYLTRANSFERASE MEASURED BY FLOW CYTOMETRIC ANALYSIS IN BLOOD AND BONE-MARROW MONONUCLEAR-CELLS

Alkyltransferase (AGT) repairs alkylation at O-6-guanine in DNA and is a major determinant of susceptibility to alkylating chemotherapeutic agents and carcinogens, Using a newly developed flow cytometry assay w ith the monoclonal anti-ACT antibody, mT3.1, we compared AGT expressio n in single-cell suspensions with standard biochemical and Western blo t assays to validate the fluorescence-activated cell sorting (FAGS) me thod and develop potential applications, From Chinese hamster ovary ce lls (CHO) transfected with human O-6-methylguanine-DNA methyltransfera se cDNA, 6 CHO-O-6-methylguanine-DNA methyltransferase clones were iso lated that expressed 0.3 to 64 fmol/mu g DNA (by biochemical assay) of human AGT, FAGS yielded a linear relationship between mean fluorescen ce intensity and both AGT activity by biochemical assay and AGT protei n by Western blot. Using this standard curve, FACS-analyzed AGT protei n content in human peripheral blood mononuclear cells (PBMCs) from nor mal donors ranged from 6.1 to 12.8 fmol/mu g DNA, similar to those obt ained by biochemical assay and Western blot, This suggests that the le vel of immunoreactive protein appears to be an accurate predictor of A GT activity in the steady state, FAGS-ACT in PBMCs from normal donors had a low index of heterogeneity within the sample, In contrast, by FA CS-AGT analysis of human bone marrow samples and granulocyte-colony-st imulating factor-mobilized PBMCs, AGT was lower and had an 8-fold high er index of heterogeneity than observed in PBMCs from normal donors, A fter treatment with O-6-benzylguanine (O-6-bG), Western and FACS-AGT d etected significant levels of AGT protein for up to 24 h, whereas bioc hemical assay showed AGT activity less than 5% of the basal level, Bec ause only the biochemical assay accurately measures net AGT activity, the AGT-FACS assay will not be useful in clinical trials to assess the efficacy of O-6-bG or other AGT inhibitors, Thus, ACT-FACS can rapidl y assess the heterogeneity of steady-state AGT in single-cell suspensi ons and may be useful for assay in lymphocytes, bone marrow cells, leu kemic myeloma plasma cells, or cells transfected with the AGT gene; We stern blot analysis is better for small samples such as tumor biopsies , whereas biochemical assay is best able to measure enzyme activity an d its inactivation by O-6-bG or other agents.