Phenylalanine Mustard-resistant Murine

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.52.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of ~-[U-'~C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of y-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of y-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-CYS-G~Y. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and LAla-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-CYS-G~Y, and L-AlaL-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-CYS-G~Y. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both y-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by yglutamylcysteine synthetase.