The First Korean Adult Case of Progressive Familial Intrahepatic Cholestasis Type 7 with Novel USP53 Splicing Variants by Next Generation Sequencing.

Progressive familial intrahepatic cholestasis (PFIC) is a group of rare genetic disorders caused by defects in biliary epithelial transporters. It mostly presents as low γ-glutamyltransferase cholestasis. Recently, USP53 has been identified as one of the novel genes associated with PFIC. Herein, we report a 21-year-old Korean male patient with a late-onset PFIC. Initial work-up, including whole genome sequencing, did not find any associated gene. However, reviewing sequencing data identified novel compound heterozygous variants in splicing site of USP53 (NM_001371395.1:c.972+3_972+6del, and c.973-1G>A). The patient's bilirubin level fluctuated during the disease course. At 4.5 years after the initial presentation, the patient's symptom and high bilirubin level were normalized after administration of high-dose ursodeoxycholic acid. Recognition of this disease entity is important for prompt diagnosis and management. USP53 is recommended for the work-up of low γ-glutamyltransferase cholestasis.

γ-Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.).

Theanine is an important secondary metabolite endowing tea with umami taste and health effects. It is essential to explore the metabolic pathway and regulatory mechanism of theanine to improve tea quality. Here, we demonstrated that the expression patterns of CsGGT2 (γ-glutamyl-transpeptidase), participated in theanine synthesis in vitro in our previous research, are significantly different in the aboveground and underground tissues of tea plants and regulated by light. Light up-regulated the expression of CsHY5, directly binding to the promoter of CsGGT2 and acting as an activator of CsGGT2, with a negative correlation with theanine accumulation. The enzyme activity assays and transient expression in Nicotiana benthamiana showed that CsGGT2, acting as bifunctional protein, synthesize and degrade theanine in vitro and in planta. The results of enzyme kinetics, Surface plasmon resonance (SPR) assays and targeted gene-silencing assays showed that CsGGT2 had a higher substrate affinity of theanine than that of ethylamine, and performed a higher theanine degradation catalytic efficiency. Therefore, light mediates the degradation of theanine in different tissues by regulating the expression of the theanine hydrolase CsGGT2 in tea plants, and these results provide new insights into the degradation of theanine mediated by light in tea plants.

γ-Glutamyl transpeptidase-activatable near-infrared nanoassembly for tumor fluorescence imaging-guided photothermal therapy.

Rationale: Precise treatment of tumors is attracting increasing attention. Molecular probes simultaneously demonstrating the diagnostic signal and pharmacological effect in response to tumor microenvironment are highly desired. γ-glutamyl transpeptidase (GGT) is a biomarker with significantly up-regulated expression in the tumor area. We developed a GGT responsive near-infrared (NIR) nanoassembly for tumor-specific fluorescence imaging-guided photothermal therapy. Methods: The GGT responsive NIR probe was constructed by conjugating GGT-specific substrate γ-glutamic acid (γ-Glu) with cyanine fluorophore (NRh-NH2) via amide reaction. The resulting NRh-G spontaneously assembled into nanoparticles (NRh-G-NPs) around 50 nm. The NPs were characterized and the properties evaluated in the presence or absence of GGT. Subsequently, we studied fluorescence imaging and photothermal therapy of NRh-G-NPs in vitro and in vivo. Results: NRh-G-NPs, upon specific reaction with GGT, turned into NRh-NH2-NPs, showing a ~180-fold fluorescence enhancement and excellent photothermal effect recovery. NRh-G-NPs could selectively light up U87MG tumor cells while their fluorescence was weak in L02 human normal liver cells. The NPs also showed excellent tumor cell ablation upon laser irradiation. After intravenous injection into tumor-bearing mice, NRh-G-NPs could arrive in the tumor area and specifically light up the tumor. Following laser irradiation, the tumor could be completely erased with no tumor reoccurrence for up to 40 days. Conclusions: NRh-G-NPs were specifically responsive to GGT overexpressed in U87MG tumor cells and selectively lit up the tumor for imaging-guided therapy. Besides, the recovery of photothermal property in the tumor area could improve cancer therapy precision and decreased side effects in normal tissues.

Recovery of Ggt7 and Ace Expressions in the Colon Alleviates Collagen-Induced Arthritis in Rats by Specific Bioactive Polysaccharide Intervention.

Rheumatoid arthritis (RA) causes swollen joints and irreversible joint damage and may even elevate cancer risks. Several bioactive nonstarch polysaccharides (NSPs) were reported to alleviate RA, but the key colonic genes accountable for this alleviation were elusive. Using collagen-induced arthritis as an RA model, colonic candidate genes related to RA were selected by transcriptome and methylome. The key genes were determined by comparing the transcriptome, methylome, and quantitative reverse transcription polymerase chain reaction profiles in RA rats with and without Lycium barbarum polysaccharides' treatment and further validated using Angelica sinensis polysaccharides and Astragalus propinquus polysaccharides for comparison. Both colonic genes γ-glutamyltransferase 7 (Ggt7) and angiotensin-I-converting enzyme (Ace) were downregulated by RA, and they were upregulated after L. barbarum polysaccharides' and A. sinensis polysaccharides' intervention that reduced the RA-caused hypermethylation status in nucleotide sites in the exon/promoter region of the two genes. However, the A. propinquus polysaccharides' intervention barely reduced the hypermethylation in the corresponding sites, failing to recover the expressions of these two genes and improve RA. Therefore, the colonic Ggt7 and Ace can be considered as key genes accountable for RA alleviation by bioactive NSP intervention. This study provides a more comprehensive insight into diet intervention to improve RA.

Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides.

The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of Escherichia coli is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. In vitro characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCE Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

An overview of biological production of L-theanine.

L-Theanine (γ-glutamylethylamide) is a unique non-protein amino acid that is naturally found in tea plants. It contributes to the umami taste and unique flavor to green tea infusion, and thus its content in tea leaves highly impacts the tea quality and price. In addition to the graceful taste, it has been proved to have many beneficial physiological effects, especially promoting relaxation and improving concentration and learning ability. Based on these promising advantages, L-theanine has been commercially developed as a valuable ingredient for use in food and beverages to improve and/or maintain human health. L-Theanine can be obtained by chemical synthesis or isolation from tea, while chemical synthesis of L-theanine is hard to be accepted by consumers and is not allowed to use in food industry, and isolation of L-theanine in high purity generally involves time-consuming, cost-ineffective, and complicated operational processes. Accordingly, the biological production of L-theanine has recently attracted much attention. Four kinds of bacterial enzymes, including L-glutamine synthetase, γ-glutamylmethylamide synthetase, γ-glutamyltranspeptidase, and L-glutaminase, have been characterized to have L-theanine-producing ability. Herein, an overview of recent studies on the biological production of L-theanine was presented.

Cloning and expression of γ-glutamyl transpeptidase and its relationship to greening in crushed garlic (Allium sativum) cloves.

BACKGROUND: Garlic greening occurs when garlic cloves are stored at low temperature, increasing 1-propenyl cysteine sulfoxide, which is induced by γ-glutamyl transpeptidase (GGT) activity. Although the metabolism of the γ-glutamyl peptide is important for the biosynthesis of green pigments in crushed garlic cloves, garlic GGT is poorly characterised. RESULTS: For the analysis of GGT at the gene level, the garlic GGT sequence was partially cloned using an onion GGT sequence. The relationship between garlic greening and related gene expressions, depending on storage condition, was investigated using reverse transcription polymerase chain reaction for garlic GGT and alliinase. Three storage conditions were set: A, storage at a constant temperature of 20 °C; B, storage at 20 °C for 3 months and then transfer to 0 °C for an additional 3 months; C, storage at 0 °C for 3 months and then transfer to 20 °C for an additional 3 months. GGT expression increased under storage condition B and decreased under storage condition C. However, alliinase expression was not affected by storage condition. CONCLUSION: Greening in crushed garlic cloves increases with increasing GGT expression at low temperature, while alliinase expression is not affected.

Inhibition of metastasis of B16F-10 melanoma cells in C57BL/6 mice by an extract of Calendula officinalis L flowers.

AIM: To determine the effect of a Calendula officinalis flower extract on lung metastasis by B16F-10 melanoma cells in C57BL/6 mice. MATERIALS AND METHODS: Male mice were injected with B16F-10 melanoma cells through the tail vein and simultaneously treated with C.officinalis flower extract. Parameters studied were lung tumor nodule count, life span of animals, gamma glutamyl transpeptidase activity, sialic acid, TNF-α, IL-1ß, IL-6, IL-2, GM-CSF, VEGF and TIMP-1 levels in serum, and lung hydroxyproline, uronic acid and hexosamine levels, as well as histopathological features. Effects of C.officinalis on the expression of various genes involved in metastasis like matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), prolyl hydoxylase, lysyl oxidase, nm23, and proinflammatory cytokines were also investigated. RESULTS: Simultaneous administration of C. officinalis extract to tumor bearing C57BL/6 mice reduced the lung tumor nodules by 74% with 43.3% increase in life span. Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and γ-glutamyl transpeptidase in the metastatic controls were found to be significantly lowered in the C. officinalis treated animals. The extract also inhibited expression of MMP-2, MMP-9, prolyl hydroxylase and lysyl oxidase and activated TIMP-1 and TIMP-2 and downregulated proinflammatory cytokines. CONCLUSIONS: The present investigation indicated antimetastatic effects of Calendula officinalis flowers through the inhibition of key enzymes involved in processes of metastasis.

Response to different oxidants of Saccharomyces cerevisiae ure2Delta mutant.

Growth of Saccharomyces cerevisiae ure2Delta mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H(2)O(2) was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Delta mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the gamma-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.

Glutamine affects glutathione recycling enzymes in a DMBA-induced breast cancer model.

Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in gamma-glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the gamma-glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of gamma-glutamyl transpeptidase (GTP), gamma-glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), gamma-glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells.

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, g-glutamyltransferase-like activity 1, slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene, and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer cells.

Purification and cloning of a gamma-glutamyl transpeptidase from onion (Allium cepa).

Gamma-glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of gamma-glutamyl linkages in gamma-glutamyl peptides and transfer of the gamma-glutamyl group to amino acids and peptides. Although plant gamma-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate gamma-glutamyl-p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion gamma-glutamyl peptide, gamma-glutamyl trans-S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme.

Protective role of vanadium on the early process of rat mammary carcinogenesis by influencing expression of metallothionein, GGT-positive foci and DNA fragmentation.

Vanadium, a dietary micronutrient, is now proving to be a promising anti-tumour agent. The present study was conducted to ascertain its anti-neoplastic potential against an experimental mammary carcinogenesis. Female Sprague-Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(alpha)anthracene (DMBA; 0.5 mg per 100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 p.p.m. was supplemented in the drinking water and given ad libitum to the experimental group immediately after the carcinogen treatment and it continued until the termination of the study (24 weeks for histological, immunological and biochemical observations and 35 weeks for morphological findings). It was found that vanadium treatment brought about substantial protection against DMBA-induced mammary carcinogenesis. This was evident from histological findings that showed substantial repair of hyperplastic lesions following supplementation of vanadium alone. There was a significant reduction in incidence (P<0.05), total number, multiplicity (P<0.01), size of palpable mammary tumours and delay in mean latency period of tumour appearance (P<0.001) following vanadium supplementation compared to the DMBA control. The immunohistochemical localization of metallothionein (a prognostic marker for breast cancer) showed reduced expression with vanadium treatment. Further, DNA fragmentation in the mammary tissue of the vanadium-treated group indicated apoptosis. In this group, vanadium also caused a significant decrease in the number (P<0.002) and focal area (P<0.05) of gamma-glutaminetranspeptidase-positive hepatic foci. The results clearly show the anti-neoplastic potential of vanadium.

Reversible skeletal abnormalities in gamma-glutamyl transpeptidase-deficient mice.

Gamma-glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the gamma-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGT-/-) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGT-/- mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the observed defects are secondary to other abnormalities. N-acetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGT-/- mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGT-/- mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGT-/- N-acetylcysteine-treated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth.

Gamma-glutamyl transpeptidase-deficient mice are resistant to the nephrotoxic effects of cisplatin.

We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.

Gamma-glutamyl transferase expression in higher-grade astrocytic glioma.

Increased expression of gamma-glutamyltransferase (GGT) has been detected in a range of human malignancies and is thought to be involved in neoplastic proliferation and treatment resistance. Since GGT expression and its role in malignant glioma biology remain largely unknown, we investigated this phenomenon by immunostaining 26 higher-grade human astrocytic gliomas (WHO grades III and IV) with a monoclonal anti-GGT-antibody (138H11). Further, human pancreatic GGT cDNA was used for liposome-mediated transfection of 9L gliosarcoma cells. GGT-expressing and control 9L cells were cultured in media containing different amounts of essential amino acids and/or cytotoxic agents. Cell viability was evaluated by microplate MTT assay. Immunohistochemical staining of tumor specimens demonstrated that GGT expression is a frequent feature of higher-grade human astrocytic gliomas, but not of normal brain tissue. Human tumors were strongly GGT-positive in 6 of 7 cases of grade III astrocytoma, and in 12 of 19 grade IV astrocytoma (glioblastoma multiforme, GBM) cases. In the cell culture model, 9L-GGT cells had a growth advantage over control cells in cysteine-deficient medium. but not in standard or glutamine-free medium. No significant difference in numbers of viable cells of either clone was found in media containing the alkylating drug BCNU (5-200 microg/ml). In conclusion, GGT is expressed in a high percentage of human WHO grade III astrocytomas and GBM, but not in normal brain tissue. This molecule seems to give neoplastic cells a moderate growth advantage under in vivo conditions.

Evidence for the pro-oxidant effect of gamma-glutamyltranspeptidase-related enzyme.

It has been previously reported that the metabolism of reduced glutathione (GSH) by gamma-glutamyltranspeptidase (GGT) in the presence of chelated metals leads to free radical generation and lipid peroxidation (LPO). The present study demonstrates for the first time that an established cell line expressing GGT-rel, a human GGT-related enzyme, metabolizes extracellular GSH to cysteinylglycine (CysGly) in a time-dependent manner when cells were incubated in a medium containing 2.5 mM GSH and 25 mM glycylglycine. Supplementation with 150-165 microM Fe(3+)-EDTA resulted in a reactive oxygen species (ROS) generation process. The resulting data showed a significantly higher level (7.6-fold) of ROS production in the GGT-rel positive cells in comparison with the GGT-rel negative control cells. CysGly and Cys, but not GSH, were responsible for the observed ROS production, as we confirmed by measuring the same process in the presence of Fe(3+)-EDTA and different thiols. A higher iron reduction and an increased LPO level determined by malondialdehyde HPLC measurement were also found in GGT-rel-overexpressing cells compared to GGT-rel negative cells. Our data clearly indicate that in the presence of iron, not only GGT, but also GGT-rel has a pro-oxidant function by generation of a reactive metabolite (CysGly) and must be taken into account as a potential physiopathological oxidation system.

gamma-glutamyl leukotrienase, a gamma-glutamyl transpeptidase gene family member, is expressed primarily in spleen.

We have recently identified a mouse enzyme termed gamma-glutamyl leukotrienase (GGL) that converts leukotriene C4 (LTC4) to leukotriene D4 (LTD4). It also cleaves some other glutathione (GSH) conjugates, but not GSH itself (Carter, B. Z., Wiseman, A. L., Orkiszewski, R., Ballard, K. D., Ou, C.-N., and Lieberman, M. W. (1997) J. Biol. Chem. 272, 12305-12310). We have now cloned a full-length mouse cDNA coding for GGL activity and the corresponding gene. GGL and gamma-glutamyl transpeptidase constitute a small gene family. The two cDNAs share a 57% nucleotide identity and 41% predicted amino acid sequence identity. Their corresponding genes have a similar intron-exon organization and are located 3 kilobases apart. A search of Genbank and reverse transcription-polymerase chain reaction analysis failed to identify additional family members. Mapping of the GGL transcription start site revealed that the GGL promoter is TATA-less but contains an initiator, a control element for transcription initiation. Northern blots for GGL expression were negative. As judged by ribonuclease protection, in situ hybridization, and measurement of enzyme activity, spleen had the highest level of GGL expression. GGL is also expressed in thymic lymphocytes, bronchiolar epithelial cells, pulmonary interstitial cells, renal proximal convoluted tubular cells, and crypt cells of the small intestine as well as in cerebral, cerebellar, and brain stem neurons but not in glial cells. GGL is widely distributed in mice, suggesting an important role for this enzyme.

Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents.

Hepatoma cell lines can be characterized by their expression of hepatocyte- and biliary-specific genes and by their response to differentiating agents in a lineage-dependent manner. These characteristics can be used to map the maturational lineage position of the cell lines. Tissue-specific gene expression and regulation by heparin, dimethylsulfoxide (DMSO), and sodium butyrate (SB) were examined in three rat hepatoma cell lines and two rat liver epithelial cell lines. Based on antigenic profiles and gene expression in serum-supplemented medium, the hepatoma cell lines could be organized in distinct categories of hepatic differentiation. All three hepatomas expressed the following five genes: gamma-glutamyl transpeptidase (GGT), glutathione-S-transferase pi (Yp), glutamine synthetase, and alpha 5 and beta 1 integrin. Cell line H4AzC2 also expressed alpha-fetoprotein (AFP), albumin. IGF II receptor, and the biliary/oval cell antigens OC.2 and OC.3, a phenotype characteristic of fetal hepatocytes. FTO-2B cells lacked AFP, OC.2, and OC.3 but expressed albumin and IGF II receptor in addition to the five commonly expressed genes, consistent with a more hepatocyte-like phenotype. Cell line H5D-7 expressed neither albumin nor the IGF II receptor, but did express OC.2, OC.3, and alpha 3 integrin in addition to the five commonly expressed genes, characteristic of biliary epithelial cells. Regulation of gene expression by heparin, DMSO, and SB was examined in cells cultured in hormonally defined medium. The patterns of regulation of AFP, albumin, GGT, and Yp were dependent upon the state of differentiation of the cell. FTO-2B cells regulated genes in a manner similar to that of E16 fetal hepatocytes, H4AzC2 regulated genes characteristic of both hepatocytic and biliary lineages, and H5D.7 regulated only biliary genes. Suppression of GGT by DMSO was uniformly observed. The three cell lines expressed equal amounts of HNF-4, but FTO-2B cells expressed more HNF-3 beta and less HNF-3 alpha, while the reverse was true of H4AzC2 and H5D.7 cells.

Expression of gamma-glutamyl transpeptidase provides tumor cells with a selective growth advantage at physiologic concentrations of cyst(e)ine.

Cells from the GGT-negative mouse hepatoma cell line, Hepa 1-6, were transfected with a human GGT cDNA and stably transformed clones were isolated. In standard tissue culture medium the GGT-positive cells and GGT-negative controls grew equally well. However, when the cysteine concentration of the medium was reduced to physiologic levels the GGT-positive cells had a growth advantage. Further investigation revealed that the medium of the GGT-negative Hepa 1-6 cells contained glutathione that had been excreted by the cells, but no glutathione was present in the medium of the GGT-positive cells. We have previously shown that expression of GGT enables cells to use extracellular glutathione as a source of cysteine (Hanigan and Ricketts, Biochem., 32:6302, 1993). These new data reveal that physiologic levels of cysteine can be limiting for cell growth and expression of GGT can provide the cells with a selective growth advantage. These data explain the observation that cells transfected with GGT grow at the same rate as the GGT-negative controls in tissue culture medium which contains a high level of cysteine, but the GGT-positive cells grow more rapidly than the GGT-negative cells when transplanted into animals (Warren et al., Proc. Soc. Exp. Biol. Med., 202:9, 1993). GGT-positive tumor cells have a selective growth advantage in vivo in comparison to GGT-negative tumor cells because they are able to use serum glutathione as a secondary source of cysteine thereby overcoming the growth restriction imposed by serum levels of cysteine.