J. Biol. Chem., Vol. 262, Issue 31, 15048-15053, Nov, 1987

Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

S Ahmad, L Okine, B Le, P Najarian and DT Vistica
Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, Maryland 20892.

Murine L1210 leukemia cells resistant to the antineoplastic agent L- phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma- 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 gamma- 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-Gly. This is supported by the observation that these preparations readily degrade L- Leu-p-nitroanilide and L-Ala-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-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L- cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase- initiated catabolism of GSH to cysteine and its reutilization by gamma- glutamylcysteine synthetase.
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