Glutamyl Hydrolase Research Articles

Overexpression of gamma glutamyl hydrolase predicts extranodal extension in squamous cell carcinoma of the oral cavity

Extranodal extension (ENE) is an important prognostic factor in oral squamous cell carcinoma (OSCC). However, ENE is only confirmed postoperatively by histologic assessment of the lymph nodes after neck dissection. Accurate identification of ENE preoperatively would help in management of OSCC. We determined the expression of molecular markers gamma glutamyl hydrolase (GGH), cyclin-dependent kinase inhibitor-3 (CDKN3), and chromobox homolog-7 (CBX7) using immunohistochemistry in OSCC clinical samples (n=35). The intensity of staining was scored using a semiquantitative index (HSCORE). The association between clinicopathologic parameters and expression of molecular markers with ENE status was analyzed using chi-square test. The number of positive nodes and the highest anatomic level of nodal involvement significantly correlated with ENE (P < .05). High GGH expression was significantly associated with ENE (P < .05), with an increased risk for ENE (odds ratio [OR] 9.9, 95% CI 1.08-91.47, P=.04), whereas no significant association was seen for CDKN3 and CBX7 expression with ENE. However, a trend toward significance was observed with a high level of CDKN3 and a low level of CBX7 expression with ENE. Gamma glutamyl hydrolase offers potential as a predictor for ENE in OSCC, whereas the role of CDKN3 and CBX7 need to be validated in a larger sample.

Rationale and preclinical development of LEAF-1401 and LEAF-1701: A novel class of potent next generation antifolates.

e14515 Background: Polyglutamation of antifolates by folylpolyglutamate synthase (FPGS) greatly enhances their activity, e.g. pentaglutamated pemetrexed is 80-fold more potent at inhibiting thymidylate synthase than pemetrexed. Polyglutamated antifolates however, do not readily cross the cell membrane, due to high negative charge and molecular mass. The resulting poor intracellular bioavailability greatly limits their therapeutic potential. Gamma glutamyl hydrolase (GGH) enzyme removes the glutamate residues from polyglutamates, which then are subject to efflux pumps. Resistance to pemetrexed has been linked to downregulation of both FPGS and folate transporters and upregulation of both GGH and the efflux pumps. We have been developing a novel class of nanoliposomal polyglutamated-antifolates including: LEAF-1401; nanoliposomal gamma-L pentaglutamated antifolate LEAF-1701; nanoliposomal alpha-L pentaglutamated antifolate These were designed to address key challenges of currently used antifolates namely: Bypassing the need for intracellular endogenous FPGS Direct delivery of the much more active polyglutamates through nanoliposome technology Minimizing toxicity to normal cells Mitigating against key antifolate resistance mechanisms such as downregulation of FPGS and folate transporters, and upregulation of GGH and efflux pumps Methods: In vitro testing in cell lines and in vivo testing in mice were performed using LEAF-1701 and LEAF-1401 in various tumor types. Results: Compared to pemetrexed: In vitro, LEAF-1701 and LEAF-1401 were more potent, in various cell lines while sparing normal neutrophils, colon and liver cells. In vivo, in H292 lung cancer xenografts, treatment with LEAF-1701 improved survival to 59 days vs 39 days for pemetrexed. In A549 lung cancer mouse orthotopic models, treatment with LEAF-1401 reduced metastatic tumor burden and prevented new metastases. In vivo, in mice doses of up to 80 mg/kg IV weekly were tolerated with little impact on blood counts and chemistry. Conclusions: LEAF-1701 and LEAF-1401 are innovative new chemical entities with promising preclinical activity and improved safety profiles and are now advancing to the clinic.

Discovering Putative Protein Targets of Small Molecules: A Study of the p53 Activator Nutlin.

Small molecule drugs bind to a pocket in disease causing target proteins based on complementarity in shape and physicochemical properties. There is a likelihood that other proteins could have binding sites that are structurally similar to the target protein. Binding to these other proteins could alter their activities leading to off target effects of the drug. One such small molecule drug Nutlin binds the protein MDM2, which is upregulated in several types of cancer and is a negative regulator of the tumor suppressor protein p53. To investigate the off target effects of Nutlin, we present here a shape-based data mining effort. We extracted the binding pocket of Nutlin from the crystal structure of Nutlin bound MDM2. We next mined the protein structural database (PDB) for putative binding pockets in other human protein structures that were similar in shape to the Nutlin pocket in MDM2 using our topology-independent structural superimposition tool CLICK. We detected 49 proteins which have binding pockets that were structurally similar to the Nutlin binding site of MDM2. All of the potential complexes were evaluated using molecular mechanics and AutoDock Vina docking scores. Further, molecular dynamics simulations were carried out on four of the predicted Nutlin-protein complexes. The binding of Nutlin to one of these proteins, gamma glutamyl hydrolase, was also experimentally validated by a thermal shift assay. These findings provide a platform for identifying potential off-target effects of existing/new drugs and also opens the possibilities for repurposing drugs/ligands.

Expression Of Gamma Glutamyl Hydrolase And Carbonic Anhydrase 9 In Oral Squamous Cell Carcinoma And Their Association With Extracapsular Spread

Introduction: Extracapsular spread (ECS) is one of the most important prognostic factors in oral squamous cell carcinoma (OSCC). Gamma Glutamyl Hydrolase (GGH) is a lysosomal enzyme which is involved in folate homeostasis. It is overexpressed in several human malignancies but its role in OSCC has never been reported. Carbonic Anhydrase 9 (CA9) is a transmembrane glycoprotein and is related to hypoxia. High expression of CA9 has been associated with poor prognosis in several tumours including OSCC. This study was aimed to investigate the expression of GGH and CA9 in OSCC and their potential use as biomarkers to predict ECS. Materials and Methods: Immunohistochemical staining with GGH and CA9 markers were performed in 35 cases of OSCC (19 with ECS and 16 without ECS) and 5 cases of normal mucosa. A semi-quantative index was used for immunoscoring. Association between the expression of these markers and ECS status were analyzed using chi-square test. Results: Immunohistochemical results indicated that GGH and CA9 were upregulated in OSCC. High GGH and CA9 expression were significantly associated with ECS (p&lt;0.05). Combination of GGH and CA9 showed greater prognostic accuracy as compared to the individual markers. Conclusion(s): Both separately and in combination GGH and CA9 offer potential as prognostic biomarkers in OSCC and was thus reliable in segregating the OSCC cases based on ECS status.

How does conserved dopamine neurotrophic factor protect against and rescue neurodegeneration of PC12 cells?

Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo, but its potential value in treating Parkinson's disease remains controversial. Here, we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells. Afterwards, conserved dopamine neurotrophic factor was administrated as a therapeutic factor, both pretreatment and posttreatment. Our results showed that (1) conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology, and attenuated alpha-synuclein accumulation in differentiated PC12 cells. (2) Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre- and posttreatment with conserved dopamine neurotrophic factor. Similarly, 26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor. (3) With regard proteolytic enzymes (specifically, glutamyl peptide hydrolase, chymotrypsin, and trypsin), glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre- and posttreatment with conserved dopamine neurotrophic factor. However, upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor. There was no change in trypsin expression. We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities, and thereby protects and rescues PC12 cells against neurodegeneration.

Pharmacogenetics of methotrexate in rheumatoid arthritis: A systematic review

Abstract Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by inflammation of multiple joints, leading to destruction of cartilage and juxta articular bone. It eventually leads to deformity, disability, and impaired quality of life. Methotrexate (MTX) has a reported response rate of 33–65%, and this variability may be explained by genetic variations (polymorphisms) in the metabolic pathway of this drug. To evaluate possible relationships between polymorphisms in the metabolic pathway and response to MTX in patients with RA. Methodology A systematic search and review of the literature was conducted. A total of 29 studies that evaluated polymorphisms in the metabolic pathway of MTX were included, due to their full text and methodological quality. Results Of the 29 studies, five were systematic reviews and/or meta-analyses, three of which clinical trials none was triple blind and only one was double-blind, six were cohort, seven were case–control, and eight cross sectional. The polymorphism identified were: methylene tetrahydrofolate reductase, dihydrofolate reductase, thymidylate synthase, 5-amino-imidazole-4-carboxamide ribonucleotide formyl transferase (AICAR formyltransferase), 5-aminoimidazole-4-carboxamide polymorphisms formyltransferase/IMP cyclohydrolase ribonucleotide (ATIC) identified conveyors attached to ATP cassette (ABC ATP-binding cassette), folylpoly-glutamate, glutamyl hydrolase, reduced folate carrier (RFC-SLC10A1). The dihydrofolate reductase and methylene tetrahydrofolate reductase polymorphism were shown to be associated with increased MTX toxicity. RFC and C677T polymorphisms are associated with better efficacy of MTX. Conclusions The polymorphisms of methylene tetrahydrofolate reductase, C677T and RFC1-G80A generate increased efficacy and toxicity in patients treated with MTX. However, for the other polymorphisms, although studies show statistically significant associations, they are not conclusive and some are contradictory. This justifies conducting multicenter studies to assess the presence and association with the effectiveness or toxicity in patients with RA treated with MTX.

Farmacogenética del metotrexato en artritis reumatoide. Revisión sistemática

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterised by inflammation of multiple joints, leading to destruction of cartilage and juxta articular bone. It eventually leads to deformity, disability, and impaired quality of life. Methotrexate (MTX), has a reported response rate of 33 to 65%, and this variability may be explained by genetic variations (polymorphisms) in the metabolic pathway of this drug.To evaluate possible relationships between polymorphisms in the metabolic pathway and response to MTX in patients with RA. MethodologyA systematic search and review of the literature was conducted. A total of 29 studies that evaluated polymorphisms in the metabolic pathway of MTX were included, due to their full text and methodological quality. ResultsOf the 29 studies, five were systematic reviews and/or meta-analyses, three of which clinical trials none was triple blind and only one was double-blind, six were cohort, seven were case-control, and eight cross sectional. The polymorphism identified were: methylene tetrahydrofolate reductase, dihydrofolate reductase, thymidylate synthase, 5-amino-imidazole-4- carboxamide ribonucleotide formyl transferase (AICAR formyltransferase), 5-aminoimidazole-4- carboxamide polymorphisms formyltransferase/IMP cyclohydrolase ribonucleotide (ATIC) identified conveyors attached to ATP cassette (ABC ATP-binding cassette), folylpoly-glutamate, glutamyl hydrolase, reduced folate carrier (RFC-SLC10A1). The dihydrofolate reductase and methylene tetrahydrofolate reductase polymorphism were shown to be associated with increased MTX toxicity. RFC and C677T polymorphisms are associated with better efficacy of MTX. ConclusionsThe polymorphisms of methylene tetrahydrofolate reductase, C677T and RFC1 - G80A generate increased efficacy and toxicity in patients treated with MTX. However, for the other polymorphisms, although studies show statistically significant associations, they are not conclusive and some are contradictory. This justifies conducting multicentre studies to assess the presence and association with the effectiveness or toxicity in patients with RA treated with MTX.

Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis

Parkinson's disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).

Fate of folates during vegetable juice processing — Deglutamylation and interconversion

A high performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (HPLC-ESI–MS/MS) was applied for quantitatively determining folate species and polyglutamyl 5-methyltetrahydrofolates (5MTHF) of various chain lengths in vegetable juices. 5MTHF polyglutamyls were the focus of this study since 5MTHF was the most abundant form in all vegetables investigated. Mono-glutamyl 5MTHF has been found to be highly bioavailable and native vegetable gamma glutamyl hydrolase (GGH) might effect this conversion. During juicing 5MTHF polyglutamyls in some vegetables were highly deglutamylated with turnip (monoglutamyl 5MTHF increased from 5.9% to 70.9%) and carrot (monoglutamyl 5MTHF increased from 2.9% to 53.4%), partially converted with broccoli (triglutamyl 5MTHF increased from 2.3% to 81.6%) and turnip greens (triglutamyl 5MTHF increased from 3.4% to 77.7%), or mostly unchanged (beet, beet greens, romaine lettuce, carrot greens). Deglutamylation of folates in turnip greens and broccoli juices terminated with 75–80% of 5MTHF species as tri-glutamyl (both Brassica species) while all other vegetables displaying GGH activity progressed to monoglutamyl 5MTHF. Total folate was mostly recovered (there are no statistical difference of total folate in juiced vegetables and unprocessed vegetables) in all vegetable juices except carrot green juice (ca. 1/3 loss). 5MTHF was the dominant folate species for all vegetables except romaine lettuce, carrot greens and turnip greens which also contained significant levels of 10-formyl species and THF. Interconversion of folates was observed during juicing usually enhancing THF levels with coincident decreases in 5MTHF and 10CHO-related species. The total folate in the whole vegetables ranged from 41 to 219μg/100 FW while ranged from 28 to 180μg/100 FW in the corresponding juice. In this study of juices from many different vegetables, beet greens and romaine lettuce yielded juices qualifying as excellent sources of folate, mostly in their monoglutamyl form, which may be more bioavailable than their polyglutamyl forms often predominating in raw and cooked vegetables.

The impact of GGH -401C > T polymorphism on cisplatin-based chemoradiotherapy response and survival in cervical cancer

AimsCervical cancer is the third most frequent cancer in women worldwide, mostly treated with cisplatin-based chemoradiotherapy. Since it is known that folate metabolism might interfere with cisplatin effectiveness, we intended to study the influence of the Gamma Glutamyl Hydrolase -401C>T polymorphism in treatment response in cervical cancer. MethodsWe retrospectively reviewed the clinical data of 167 patients with bulky cervical cancer submitted to cisplatin-based chemoradiotherapy. The genotypes of GGH -401C>T SNP were determined by real-time PCR and statistical analysis was performed by χ2 test and survival analysis. ResultsThe genotypes of GGH-401C>T were significantly associated with the response to platinum-based chemoradiotherapy. Treatment response was higher in patients carrying the CC genotype, who presented a significant increased chance of treatment response (survival time in months/genotype: 91 for CC Vs 72 for CT/TT; p=0.035, log rank test). A Cox regression analysis accordingly showed that the presence of the T allele was significantly linked to a worse treatment response (HR=3.036; CI 95% 1.032-8.934, p=0.044). ConclusionsThe results of our study suggested the potential interest of GGH -401C>T as a predictive factor of the outcome of cervical carcinoma treated with cisplatin-based chemoradiotherapy.

Association Between Polymorphisms of Dihydrofolate Reductase and Gamma Glutamyl Hydrolase Genes and Toxicity of High Dose Methotrexate in Children with Acute Lymphoblastic Leukemia

Methotrexate (MTX) is an important drug for the treatment of childhood acute lymphoblastic leukemia (ALL). However, related toxicity occurs in many organs which may cause interruption of treatment, morbidity, and mortality. Single nucleotide polymorphisms (SNPs) of dihydrofolate reductase (DHFR) and gamma glutamyl hydrolase (GGH) are known to alter their enzymatic activity and thus affect the metabolism of MTX and influence the effectiveness. Therefore, we hypothesized that genetic variations of DHFR and GGH genes may influence the risk of toxicity after high dose MTX. The study population comprised of 105 children with ALL who were treated according to the modified St Jude Total XV protocol. The patients received 2.5 or 5 g/m2 of MTX for 5 doses during the consolidation phase. Genotyping of DHFR 829C>T and GGH -401C>T was performed using a polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). The GGH-401CT and TT genotypes were associated with increased risk of leukopenia and thrombocytopenia after high dose MTX (OR 2.97, 95%CI; 1.24-7.13 and OR 4.02, 95%CI; 1.58-10.26). DHFR 829C>T was not associated with toxicity. In conclusion, the GGH -401CT and TT genotypes were found to increase the risk of severe leukopenia and thrombocytopenia after exposure to high dose MTX for childhood ALL therapy.

A comparative proteomic analysis of the early response to compatible symbiotic bacteria in the roots of a supernodulating soybean variety

To reveal the processes involved in the early stages of symbiosis between soybean plants and root nodule bacteria, we conducted a proteomic analysis of the response to bacterial inoculation in the roots of supernodulating (En-b0-1) and non-nodulating (En1282) varieties, and their parental normal-nodulating variety (Enrei). A total of 56 proteins were identified from 48 differentially expressed protein spots in normal-nodulating variety after bacterial inoculation. Among 56 proteins, metabolism- and energy production-related proteins were upregulated in supernodulating and downregulated in non-nodulating varieties compared to normal-nodulating variety. The supernodulating and non-nodulating varieties responded oppositely to bacterial inoculation with respect to the expression of 11 proteins. Seven proteins of these proteins was downregulated in supernodulating varieties compared to non-nodulating variety, but expression of proteasome subunit alpha type 6, gamma glutamyl hydrolase, glucan endo-1,3-beta glucosidase, and nodulin 35 was upregulated. The expression of seven proteins mirrored the degree of nodule formation. At the transcript level, expression of stem 31kDa glycoprotein, leucine aminopeptidase, phosphoglucomutase, and peroxidase was downregulated in the supernodulating variety compared to the non-nodulating variety, and their expression in the normal-nodulating variety was intermediate. These results suggest that suppression of the autoregulatory mechanism in the supernodulating variety might be due to negative regulation of defense and signal transduction-related processes.

Abstract 1596: Effects of folylpolyglutamyl synthase and γ-glutamyl hydrolase modulation on DNA methylation

Abstract Background: Folate mediates the transfer of one-carbon units for the provision of S-adenosylmethionine, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNA methyltransferase (DNMT). Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis. DNA methylation and DNMT also are potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. Intracellular folate homeostasis is maintained by folylpolyglutamate synthase (FPGS) that facilitate intracellular retention of folate by polyglutamylation and by γ -glutamyl hydrolase (GGH) that catalyzes the hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating export of folate out of the cell. We investigated whether GGH and FPGS modulation would affect genomic DNA methylation and DNMT activity. Methods: We generated an in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells by stably transfecting the cells with the sense GGH cDNA or GGH-targeted siRNA, respectively. An in vitro model of FPGS overexpression and inhibition in HCT116 cells was generated by stably transfecting the cells with the sense or antisense FPGS cDNA, respectively. Similarly, MDA-MB-435 cells were transfected with the sense FPGS cDNA or FPGS-targeted siRNA, respectively, to generate an in vitro model of FPGS overexpression and inhibition. Genomic DNA methylation and DNMT activity were determined. Results: Functionally significant GGH/FPGS overexpression and inhibition were confirmed by GGH and FPGS protein expression, GGH and FPGS activity, and total and long-chain polyglutamylated intracellular folate concentrations. In both cell lines, GGH overexpression was associated with significantly lower genomic DNA methylation and DNMT activity than controls, while GGH inhibition was associated with significantly higher genomic DNA methylation and DNMT activity than controls (P&amp;lt;0.05). FPGS overexpression was associated with lower genomic DNA methylation and DNMT activity than controls in HCT116 cells while it was associated with higher genomic DNA methylation and DNMT activity than controls in MDA-MB-435 cells (P&amp;lt;0.05). FPGS inhibition was associated with higher genomic DNA methylation and lower DNMT activity compared with controls in HCT116 cells (P&amp;lt;0.05) whereas it did not affect genomic DNA methylation in MDA-MB-435 cells. Conclusions: Our data indicate that GGH and FPGS modulation can affect genomic DNA methylation and DNMT activity of colon and breast cancer cells. Studies are underway to determine whether these altered DNA methylation and DNMT activity influence chemosensitivity of these cancer cells to 5-fluorouracil and antifolates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1596.

Epigenetic Regulation of Human γ-Glutamyl Hydrolase Activity in Acute Lymphoblastic Leukemia Cells

Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.

From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates

• Are multiple intracellular targets better than one? Does inhibition of dihydrofolate reductase (DHFR), thymidylate synthase (TS) or enzymes of the purine biosynthetic pathway confer an advantage? Alternatively, is inhibition of purine biosynthesis a safety liability rather than an advantage? • Can a lipophilic molecule rather than one that uses the folate carrier mechanisms, i.e. the folate binding protein (FBP) and or the reduced folate carrier (RFC), circumvent intrinsic or acquired resistance? • Can tumor selectivity be increased by exploiting knowledge of different patterns of RFC versus FBP expression or of variations in FBP isoform expression? • How does a patient’s functional folate status or supplemental folic acid modulate efficacy and or toxicity? • Does an antifolate that is a good substrate for the intracellular enzyme folylpolyglutamate synthase (FPGS) have a selective advantage? Does polyglutamation of the antifolate confer a therapeutic advantage because it results in increased potency against the target enzyme, and increased polarity leading to increased cellular retention? Alternatively, is it a disadvantage with respect to safety of the molecule because of prolonged intracellular retention times or the potential for resistance? • Does an increase in the potency of binding to the target enzyme confer a therapeutic advantage? • Resistance mechanisms include decreased cellular accumulation due to impaired transport, decreased retention inside the cell as a consequence of a lack of polyglutamate formation, or an increase in gamma glutamyl hydrolase (GGH) activity, an increase in expression of the target enzyme, or a mutation in the target enzyme. Which of these is clinically relevant, and how does knowledge of these resistance mechanisms aid selection of the appropriate antifolate for treatment of an individual patient? • How do pharmacokinetic variables contribute to safety and efficacy? Do the newer antifolates have fewer propensities for drug-drug interactions? • Is there an intrinsic limit to the clinical activity of an antifolate as a single agent? Can antitumor activity be increased, or can an increase in activity only be achieved by combining antifolates with other anticancer drugs (either other antifolates which target different folate requiring enzymes or compounds with different mechanisms of action)? Similarly, can the spectrum of clinical activity be increased? • Can patients whose tumors have a specific set of molecular properties be identified prospectively, and matched with the pharmacological properties of an agent, i.e. can therapy of individual patients be tailored? • Can toxicity be decreased and quality of life improved? • Can patients at risk of serious toxicity to a given agent be identified before or during therapy and managed in the light of that knowledge? • Are there mechanisms for rescuing patients from toxicity, and not from antitumor activity? • What improvements can be made in the administration schedule and therefore convenience to patients?

Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation.

The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.

Glutamyl hydrolase and the multitargeted antifolate LY231514.

To examine the activity of glutamyl hydrolase (GH) on the poly-gamma-glutamates of multitargeted antifolate (MTA) (LY231514) and the effect of enhanced GH on the pharmacological activity of MTA. Expressed and purified GH were used to study the enzymatic cleavage of MTA poly-gamma-glutamates and wild-type and GH-enhanced H35 hepatoma cell lines to evaluate growth inhibition. MTA tri- and penta-gamma-glutamates were good substrates for human GH, having higher rates than MTX tri- and penta-gamma-glutamates. Preferential hydrolysis with human enzyme occurred at the two gamma-glutamyl bonds at the carboxyl end of the molecule, whereas the rat enzyme preferred the innermost gamma-linkage. Incubation of rat H35 hepatoma cell lines with MTA resulted in the intracellular accumulation of primarily tetra-, penta-, and hexa-gamma-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. MTA poly-gamma-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-gamma-glutamates of MTA inhibiting TS as the primary target. Further evidence for the inverse relationship of GH to classical antifolate pharmacological activity is established.

Purification and partial characterization of an ATP-hydrolyzing serine protease from lettuce leaves

Abstract A novel ATP-hydrolyzing protease has been purified from lettuce leaves by combination of (NH 4 ) 2 SO 4 fractionation, gel filtration and anionic exchange chromatography. The purified enzyme is made up of a single subunit with an apparent molecular weight of 40 000, even though a possible higher molecular organization might occur in vivo induced by the presence of ATP or protease's substrate. The lettuce protease showed caseinolytic, trypsin-like and, to a smaller extent, peptidyl glutamyl hydrolase activities. It is a serine protease as both peptidase activities are highly sensitive to tosyl- l -lysinechloromethylketone (TLCK) and leupeptin. The trypsin-like activity of the enzyme was not affected by MgATP complex or ATP alone. Peptidyl glutamyl hydrolase activity, instead, and ATP hydrolysis were strictly correlated as incubation of the enzyme with MgATP, but not with ATP alone, stimulated the peptidase activity of the enzyme, while peptide substrate as well as TLCK enhanced ATPase activity. Moreover, the ATPase inhibitor vanadate, which also blocked the peptidyl glutamyl hydrolase activity, caused a strong activation of the trypsin-like activity of the enzyme. Therefore, these studies could indicate the existence of multiple functional states of the enzyme achieved in vivo by ATP hydrolysis. The cytosolic localization of the enzyme is finally discussed.

Competitive inhibition of a glutamate carboxypeptidase by phosphonamidothionate derivatives of glutamic acid

Several N-thiophosphonyl-glutamates were found to be potent competitive inhibitors of a zinc-dependent glutamyl hydrolase, carboxypeptidase G (CPG). Weak inhibition exhibited by an analogous N-phosphonyl-glutamate suggests that the enhanced potency of the phosphonamidothioates is due to the presence of their sulfur ligand and its favorable interactions with active site features, presumably zinc(II).

Identification, Cloning, and Sequencing of a cDNA Coding for Rat -Glutamyl Hydrolase

Purified gamma-glutamyl hydrolase secreted from rat H35 hepatoma cells has been characterized as a diffuse band of 55 kDa on SDS-polyacrylamide gel electrophoresis that is converted to bands of 35 and 33 kDa after enzymatic removal of N-linked carbohydrate. Polyclonal antibodies against 55-kDa gamma-glutamyl hydrolase captured the enzyme activity and recognized the glycosylated and both deglycosylated forms of gamma-glutamyl hydrolase. A complete cDNA sequence of gamma-glutamyl hydrolase was obtained using degenerate oligonucleotides derived from peptide sequences, screening of a rat hepatoma cDNA library, and reverse transcription polymerase chain reaction. Based upon the deduced amino acid sequence the peptide component of gamma-glutamyl hydrolase had a molecular weight of 33,400. The results of amino acid analysis of the purified protein agreed with the deduced amino acid sequence in which there are seven potential asparagine-containing glycosylation sites.