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-Glutamyl Hydrolase from Human Sarcoma HT-1080 Cells:
Characterization and Inhibition by Glutamine Antagonists
Program of Molecular Pharmacology and Therapeutics, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
Elevated 
-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 
2000-fold) displayed
exopeptidase activity in cleaving successive end-terminal -glutamyl
groups from poly-L--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 µM) and
[3H]MTX for 24 hr resulted in modest elevations of the
long-chain -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.
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