Abstract
Purpose: To examine the activity of glutamyl hydrolase (GH) on the poly-γ-glutamates of multitargeted antifolate (MTA) (LY231514) and the effect of enhanced GH on the pharmacological activity of MTA. Methods: Expressed and purified GH were used to study the enzymatic cleavage of MTA poly-γ-glutamates and wild-type and GH-enhanced H35 hepatoma cell lines to evaluate growth inhibition. Results: MTA tri- and penta-γ-glutamates were good substrates for human GH, having higher rates than MTX tri- and penta-γ-glutamates. Preferential hydrolysis with human enzyme occurred at the two γ-glutamyl bonds at the carboxyl end of the molecule, whereas the rat enzyme preferred the innermost γ-linkage. Incubation of rat H35 hepatoma cell lines with MTA resulted in the intracellular accumulation of primarily tetra-, penta-, and hexa-γ-glutamate. The formation of these were markedly reduced in H35D cells, which is a line resistant to antifolates chiefly through enhanced cellular levels of GH activity. Conclusions: MTA poly-γ-glutamates are effective substrates for GH and their pharmacological effectiveness bears an inverse relationship to cellular GH activity. This observation, along with enhanced resistance to MTA of thymidylate synthase-amplified cells, substantiates the importance of the poly-γ-glutamates of MTA inhibiting TS as the primary target. Further evidence for the inverse relationship of GH to classical antifolate pharmacological activity is established.
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Received: 10 November 1998 / Accepted: 30 March 1999
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Rhee, M., Ryan, T. & Galivan, J. Glutamyl hydrolase and the multitargeted antifolate LY231514. Cancer Chemother Pharmacol 44, 427–432 (1999). https://doi.org/10.1007/s002800051000
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DOI: https://doi.org/10.1007/s002800051000