Myung S. Rhee

Glutamyl hydrolase: pharmacological role and enzymatic characterization

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

Serum Factor from Patients with Chronic Renal Failure Enhances Polymorphonuclear Leukocyte Oxidative Metabolism

Sera from patients with chronic renal failure (CRF) contain a factor(s) which enhances the oxidative metabolism of polymorphonuclear leukocytes (PMN) as assessed by chemiluminescence (CL), superoxide anion generation, and hexose monophosphate shunt activity. PMN oxidative metabolic activity was higher in CRF sera than in sera from hospitalized patients with normal renal function or in sera from normal healthy subjects. The enhancement occurred regardless of whether PMN were unstimulated or were stimulated by a nonspecific soluble membrane stimulant (phorbol myristate acetate), or by opsonized Candida albicans. The enhanced CL was significantly reduced in their sera after normal renal function was restored with successful renal transplantation. This CL-enhancing factor was also detected in dialysate fluids from CRF patients and in urine from normal healthy subjects. When serum, urine, dialysate fluids of these CRF patients were fractionated by Sephadex G-25 column chromatography, the specific fraction responsible for enhanced CL was found in the molecular weight range less than 1,000 daltons, and is an ethanol extractable substance with natural fluorescence. Our findings suggest that the enhanced PMN stimulatory activity in CRF serum is specifically associated with renal dysfunction and can be useful, along with other conventional parameters, for monitoring the progression of CRF.

Glutamyl hydrolase and the multitargeted antifolate LY231514

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.

Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: Evidence for multiple pathways of hydroxyvitamin K formation☆

Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.

The role of folylpolyglutamate synthetase and γ-glutamyl hydrolase in altering cellular folyl- and antifolylpolyglutamates

The metabolic products of methotrexate differ in hepatocytes and hepatoma cells; the primary products are 7-hydroxymethotrexate in hepatocytes and the polyglutamate derivatives in hepatoma cells. The rate of glutamylation is approximately 5-fold greater in hepatoma cells than hepatocytes under comparable conditions and the polyglutamate products in the hepatoma cells consist of longer chain length tri- and tetraglutamate derivatives, whereas the hepatocytes favor the diglutamate derivative. Extracts of cultured hepatocytes have approximately half the folylpolyglutamate synthetase and three times as much gamma-glutamyl hydrolase as the hepatoma cells. A further difference is that methionine inhibits glutamylation in hepatocytes whereas it can cause a marked stimulation (five- to seven-fold) in hepatoma cells. The rate of glutamylation of methotrexate in intact hepatoma cells is strongly dependent on the culture conditions and can be enhanced as much as five-fold in dividing cultures or by removing folates from the cells. These changes may be in part caused by the fact that the folylpolyglutamate synthetase is nearly doubled in both growing cultures and in folate lacking cells. The treatment of human liver in vivo or rodent hepatic cells in vivo and in vitro with methotrexate causes a reduction in the folate pools. Data presented in these studies show that the activity of gamma-glutamyl hydrolase in cells can be enhanced by prior exposure to methotrexate. Thus, methotrexate could cause a loss in cellular folylpolyglutamates by enhancing their rate of cleavage to folylmonoglutamates.

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor

AG-331 {N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino-benz[cd]indole glucuronate} is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects on purine incorporation. In addition, H35 cells treated with 10 μM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breastcancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated.

A comparison of HPLC and ternary complex-based assays of tissue reduced folates

Abstract Two different analytical methods were used to estimate reduced folates from the same cultured hepatoma cell preparation. A method based on HPLC separation of reduced folate pools in cells prelabelled with [3H]-folic acid was compared to a method based upon quantitative assessment of bound radioactive ligand by gel filtration after entrapment of one reduced folate, methylenetetrahydrofolate, into a stable ternary complex with thymidylate synthase and [3H]-fluorodeoxyuridine monophosphate. In this second method additional folate pools were measured following enzymatic conversion to methylenetetrahydrofolate. Results show completely satisfactory agreement between the two methods both in terms of total folate content and in levels of individual folate pools.

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.

Effect of a Novel Antifolate, Nα-(4-Amino-4-Deoxypteroyl)-Nδ-Hemiphthaloyl-L-Ornithbsfe (PT523), On Growth of H35 Rat Hepatoma and HEPG2 Human Hepatoma Cells

Structural modifications of folic acid have generated various derivatives that act at different target sites. Well-known examples include the dihydrofolate reductase (DHFR) inhibitor methotrexate (MTX),1–3 the thymidylate synthase inhibitor 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid,4,5 and the glycinamide ribonucleotide formyltransferase inhibitor 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.6,7 More recently, folate analogues containing ornithine in place of glutamate have been synthesized as inhibitors of folylpolyglutamate synthetase.8,9 These ornithine-containing analogues showed weaker activity than the corresponding glutamate-containing analogues on cell growth presumably because of inefficient transport across the cell membrane.9,10 In contrast, a novel derivative of an ornithine-containing antifolate, Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-Lornithine (PT523), exhibits potent in vitro and in vivo antitumor activity.11

Phagocytosis, Chemoluminescence, and Intracellular Killing of Fungi by Phagocytes from Subjects with Deficiency of the Second Component of Complement

The capacity of phagocytes from animals or humans with complement component deficiency to ingest and kill Candida albicans has been much disputed. We show that peripheral blood polymorphonuclear leukocytes and mononuclear phagocytes from subjects with hereditary C2 deficiency (C2D) ingested C. albicans or Saccharomyces cerevisiae at an abnormally slow rate. After preincubating C. albicans in C2D plasma, the slow rate of phagocytosis was corrected and subsequent intracellular killing of C. Albicans was normal. A normal number of C2D phagocytes reduced nitroblue tetrazolium after stimulation with either phorbol myristate acetate or ingestion of C. albicans. The rate at which chemoluminescence was generated in response to C. albicans was abnormally slow, but peak chemoluminescence produced by C2D phagocytes in response to C. albicans was normal.