γ‐glutamyl transpeptidase (γ‐GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents

Abstract

Previous studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L-phenylalanine mustard (L-PAM) and other alkylating agents is accompanied by a two-to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of γ-glutamyl transpeptidase (γ-GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of γ-GT is elevated two-to threefold in L-PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of γ-GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the γ-glutamyl donor (L-γ-glutamyl-p-nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two-to threefold in L-PAM resistant tumor cells. Investigation into the role of γ-GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two-fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.