gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.
Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.
Waltham
M C
MC
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Li
W W
WW
Gritsman
H
H
Tong
W P
WP
Bertino
J R
JR
D001377:Azaserine / Q000494:pharmacology; D003980:Diazooxonorleucine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005973:Glutamine / Q000037:antagonists & inhibitors*; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D012509:Sarcoma / Q000201:enzymology*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007555:Isoxazoles; D003980:Diazooxonorleucine; D005973:Glutamine; D001377:Azaserine; D011623:gamma-Glutamyl Hydrolase; C006303:acivicin; D008727:Methotrexate