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Gamma-Glutamyl transpeptidase expression in Ewing's sarcoma cells: up-regulation by interferons.

ABSTRACT: The genetic hallmark of Ewing's sarcoma family of tumours (ET) is the presence of the translocation t(11;22)(q24;q12), which creates the ET fusion gene, leading to cellular transformation. Five human gamma-glutamyl transpeptidase (gamma-GT) genes are located near the chromosomal translocation in ET. gamma-GT is a major enzyme involved in glutathione homoeostasis. Five human cell lines representative of primary or metastatic tumours were investigated to study whether gamma-GT alterations could occur at the chromosomal breaks and rearrangements in ET. As shown by enzymic assays and FACS analyses, all ET cell lines consistently expressed a functional gamma-GT which however did not discriminate steps of ET progression. As shown previously [Sancéau, Hiscott, Delattre and Wietzerbin (2000) Oncogene 19, 3372-3383], ET cells respond to the antiproliferative effects of interferons (IFNs) type I (alpha and beta) and to a much less degree to IFN type II (gamma). IFN-alpha and -beta arrested cells in the S-phase of the cell cycle. We found an enhancement of gamma-GT mRNA species with IFN-alpha and -beta by reverse transcriptase-PCR analyses. This is reflected by up-regulation of gamma-GT protein, which coincides with the increase in gamma-GT-specific enzymic activity. Similarly, IFNs up-regulate the levels of gamma-GT in another IFN-responsive B cell line. Whether this up-regulation of gamma-GT by IFNs is of physiological relevance to cell behaviour remains to be studied.

SUBMITTER: Bouman L

PROVIDER: S-EPMC1222621 | biostudies-other | 2002 Jun

REPOSITORIES: biostudies-other

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Project description:PurposeGamma-glutamyl transpeptidase (GGT) family genes play crucial roles in the formation and progression of several solid tumors. However, the expression patterns and the prognostic significance of GGT members in hepatocellular carcinoma (HCC) remain unknown. This study was designed to determine the expression profiles of GGT family members in HCC and validate the prognostic value of serum GGT protein in patients with HCC.MethodWe comprehensively searched public resources based on the LIHC dataset to determine the expression patterns, prognostic significance, DNA methylation status, immune infiltration, and biological pathways of GGT family genes in HCC. Subsequently, we validated the prognostic value of serum GGT protein in 85 patients with early-stage HCC subjected to curative hepatectomy from the Renmin Hospital of Wuhan University.ResultsExcept for GGT1, other GGT family members (GGT5, GGT6, and GGT7) were found to be differentially expressed in primary HCC samples (N = 371) and normal control tissues (N = 50). Furthermore, a positive relationship was not only observed between GGT1 and GGT5 (Spearman coefficient: 0.24, P = 5.143 × 10-6) but also between GGT5 and GGT6 (Spearman coefficient: 0.38, P = 1.24 × 10-13). The expression of GGT1, GGT5, and GGT7 was correlated with overall survival (OS), and GGT7 was associated with disease-free survival (DFS) in patients with HCC. Negative associations between DNA methylation and expression of mRNA were observed for GGT1 (Spearman coefficient: -0.38, P = 6.24e-14), GGT6 (Spearman coefficient: -0.29, P = 1.23e-8), and GGT7 (Spearman coefficient: -0.34, P=6.7e-11). GGT family genes were well correlated with the infiltration levels of immune cells in HCC, especially CD4+ T cells, macrophages, and dendritic cells. Finally, when validated with clinical data from the Renmin cohort, a high expression of serum GGT protein was identified as a strong prognostic element of unfavorable OS (HR = 3.114, P = 0.025), but not of DFS (HR = 1.198, P = 0.05) in patients with HCC subjected to curative hepatectomy.ConclusionTo our knowledge, this is the first comprehensive analysis of the expression patterns and clinical value of GGT family genes in patients with HCC. Our study laid the foundation for the clinical application of the GGT protein in the survival assessment of patients with HCC.

Project description:Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.

Project description:BackgroundCholestatic liver diseases exhibit higher levels of serum γ-glutamyl transpeptidase (GGT) and incidence of secondary osteoporosis. GGT has been identified as a novel bone-resorbing factor that stimulates osteoclast formation. The aim of this study was to elucidate the interaction of elevated GGT levels and cholestatic liver disease-induced bone loss.MethodsWistar rats were divided into three groups: sham-operated control (SO) rats, bile duct ligation (BDL) rats, and anti-GGT antibody-treated BDL rats (AGT). Serum GGT level was measured. Bone mineral density (BMD) was analyzed by dual-energy X-ray absorptiometry. Bone morphometric parameters and microarchitectural properties were determined by micro-computed tomography and histomorphometry of the distal metaphysis of femurs. Alterations of bone metabolism-related factors were evaluated by cytokine array. Effects of GGT on osteoblasts or stromal cells were evaluated by RT-PCR, enzyme activity, and mineralization ability.ResultsSerum levels of GGT were significantly elevated in the BDL-group. In the BDL group, BMD, bone mass percentage, and osteoblast number were significantly decreased, whereas osteoclast number was significantly increased. These alterations were markedly attenuated in the AGT group. The mRNA levels of vascular endothelial growth factor-A, LPS-induced CXC chemokine, monocyte chemoattractant protein-1, tumor necrosis factor-α interleukin-1β and receptor activator of nuclear factor-kappa B ligand were upregulated, and those of interferon-γ and osteoprotegerin were downregulated in the GGT-treated stromal cells. Furthermore, GGT inhibited mineral nodule formation and expression of alkaline phosphatase and bone sialo-protein in osteoblastic cells.ConclusionOur results indicate that elevated GGT level is involved in hepatic osteodystrophy through secretion of bone resorbing factor from GGT-stimulated osteoblasts/bone marrow stromal cells. In addition, GGT also possesses suppressive effects on bone formation. Managing elevated GGT levels by anti-GGT antibody may become a novel therapeutic agent for hepatic osteodystrophy in chronic liver diseases.

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Gamma-Glutamyl transpeptidase expression in Ewing's sarcoma cells: up-regulation by interferons.

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