Search

Background Glutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells. Methods Microarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G. Results MiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression. Conclusions Glutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.

Liver-type glutaminase (LGA) is a glutaminase isoform that has been implicated in transcription modulation. LGA mRNA is absent from postoperative samples of primary gliomas and is low in cultured astrocytes. In this study, stable transfection of T98G cells with a vector carrying human LGA sequence increased the expression of LGA mRNA and protein, and the ability of the cells to degrade glutamine (Gln), as manifested by a three-fold reduction of their steady-state Gln content and a 2.5-fold increase of their glutamate (Glu) content. The transfected cells (TLGA cells) showed a 40% decrease of cell survival as assessed by colony formation, well correlated with significant reduction of mitochondrial activity as demonstrated with MTT test. Also, a 45% reduction of cell migration and a 47% decrease of proliferation index (Ki67 immunostaining) were found as compared with sham-transfected cells. Microarray analysis, which included over 47,000 transcripts, revealed a significantly altered expression of 85 genes in TLGA, but not in sham-transfected or control cells (P < 0.005). Microarray data were confirmed with real-time PCR analysis for eight genes potentially relevant to malignancy: S100A16, CAPN2, FNDC3B, DYNC1LI1, TIMP4, MGMT, ADM, and TIMP1. Of these changes, decreased expression of S100A16 and MGMT can be best reconciled with the current views on the role of their protein products in glioma malignancy. Malignancy-reducing effect of newly inserted LGA mRNA in glioblastoma cells can be reconciled with a hypothesis that absence of such a modulatory mechanism in glia-derived tumors deprived of LGA mRNA may facilitate some aspects of their progression.

Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S(0.5) values of 32 and 64 mM for high and low P(i) concentrations, respectively. Whereas the protein showed a low P(i) dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes.

Metabolism is fundamental to cell survival, growth, and behavior. Tumor cells have an enhanced demand for nutrients which provide biosynthetic building blocks and cellular energy to sustain their proliferative status. Altered metabolism is a hallmark of cancer. The term metabolic reprogramming has been coined to describe the whole range of metabolic abnormalities accompanying tumorigenesis and metastasis. Increased glutamine uptake and glutaminolysis are key metabolic traits that have been consistently found on a wide range of human and experimental cancers. Glutaminase proteins control the first step in the glutaminolytic process: the conversion of glutamine to glutamate and ammonium ions. Glutaminase expression has been correlated with malignancy and growth rate on a great variety of tumors. Recent works are now starting to uncover the differential expression of glutaminase isoenzymes and their regulation by oncogenes and tumor suppressor genes. In parallel, glutaminase isoforms are attracting great interest as novel cancer chemotherapeutic targets. The focus of this chapter will highlight the role of glutamine addiction in tumor biology with particular emphasis on the interaction between the host and the tumor.

Glutaminase is expressed in most mammalian tissues and cancer cells, but recent studies are now revealing a considerably degree of complexity in its pattern of expression and functional regulation. Novel transcript variants of the mammalian glutaminase Gls2 gene have been recently found and characterized in brain. Co-expression of different isoforms in the same cell type would allow cells to fine-tune their Gln/Glu levels under a wide range of metabolic states. Moreover, the discovery of protein interacting partners and novel subcellular localizations, for example nucleocytoplasmic in neurons and astrocytes, strongly suggest non-neurotransmission roles for Gls2 isoforms associated with transcriptional regulation and cellular differentiation. Of note, Gls isoforms have been considered as an important trophic factor for neuronal differentiation and postnatal development of brain regions. On the other hand, glutaminases are taking center stage in tumor biology as new therapeutic targets to inhibit metabolic reprogramming of cancer cells. Interestingly, glutaminase isoenzymes play seemingly opposing roles in cancer cell growth and proliferation; this issue will be also succinctly discussed with special emphasis on brain tumors.

Tumor cells expressing antisense glutaminase RNA show a drastic inhibition of glutaminase activity and they acquire a more differentiated phenotype. We have studied the expression of Sp1 and Sp3 transcription factors in both Ehrlich tumor cells and their derivative 0.28AS-2 antisense glutaminase expressing cells. The expression of phosphorylated Sp1 in 0.28AS-2 cells was 3-fold the expression in EATC. Full length Sp3 was also incremented in 0.28AS-2 cells. Sp1 and Sp3 binding to a consensus Sp1 probe was higher in 0.28AS-2 nuclear extracts, as determined by supershift assays. Sp1-DNA binding was inhibited by phosphatase treatment, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. The Sp1 and Sp3 DNA binding found in 0.28AS-2 cells was also correlated with an increased Sp1 activity, as shown in transient transfections assays carried out with a luciferase reporter plasmid. Incubation of Ehrlich tumor cells with the differentiation agent PMA could not totally reproduce the Sp1/Sp3 changes observed in 0.28AS-2 cells. However, it was demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. In conclusion, the antisense inhibition of glutaminase leads to an increased expression of phosphorylated Sp1 and that correlates with an increase in Sp1 activity.

Glutamine is an essential amino acid in cancer cells and is required for the growth of many other cell types. Glutaminase activity is positively correlated with malignancy in tumours and with growth rate in normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen peroxide. It is shown that Ehrlich ascites tumour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathione than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apoptotic cells and are more sensitive to both methotrexate and hydrogen peroxide toxicity than normal cells. Taken together, these results provide insights into the role of glutaminase in apoptosis by demonstrating that the expression of antisense mRNA for glutaminase alters apoptosis and glutathione antioxidant capacity.

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-alpha-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors.

Phosphate-activated glutaminase has a critical role in tumours and rapidly dividing cells and its activity is correlated with malignancy. Ehrlich ascites tumour cells transfected with the pcDNA3 vector containing an antisense segment (0.28 kb) of rat kidney glutaminase showed impairment in the growth rate and plating efficiency, as well as a shortage in the glutaminase protein and activity. The C-terminal segment used is well conserved in all glutaminase sequences known. The transfected cells, named 0.28AS-2, displayed remarkable changes in their morphology compared with the parental cell line. The 0.28AS-2 cells also lost their tumourigenic capacity in vivo. Control mice developed an ascitic tumour, with a lifespan of 16±1 days, when inoculated with 107 cells/mouse; on the contrary, animals inoculated with transfected cells up to 2.5 times the cell numbers of control mice did not develop tumours and behaved as healthy animals. The ability to revert the transformed phenotype of antisense-transfected cells confirms the relevance of glutaminase in the transformation process and could provide new ways for the study of gene therapy.

Mitochondrial glutaminase (GA) plays an essential role in cancer cell metabolism, contributing to biosynthesis, bioenergetics, and redox balance. Humans contain several GA isozymes encoded by the GLS and GLS2 genes, but the specific roles of each in cancer metabolism are still unclear. In this study, glioma SFxL and LN229 cells with silenced isoenzyme glutaminase KGA (encoded by GLS) showed lower survival ratios and a reduced GSH-dependent antioxidant capacity. These GLS-silenced cells also demonstrated induction of apoptosis indicated by enhanced annexin V binding capacity and caspase 3 activity. GLS silencing was associated with decreased mitochondrial membrane potential (ΔΨm) (JC-1 dye test), indicating that apoptosis was mediated by mitochondrial dysfunction. Similar observations were made in T98 glioma cells overexpressing glutaminase isoenzyme GAB, encoded by GLS2, though some characteristics (GSH/GSSG ratio) were different in the differently treated cell lines. Thus, control of GA isoenzyme expression may prove to be a key tool to alter both metabolic and oxidative stress in cancer therapy. Interestingly, reactive oxygen species (ROS) generation by treatment with oxidizing agents: arsenic trioxide or hydrogen peroxide, synergizes with either KGA silencing or GAB overexpression to suppress malignant properties of glioma cells, including the reduction of cellular motility. Of note, negative modulation of GLS isoforms or GAB overexpression evoked lower c-myc and bcl-2 expression, as well as higher pro-apoptotic bid expression. Combination of modulation of GA expression and treatment with oxidizing agents may become a therapeutic strategy for intractable cancers and provides a multi-angle evaluation system for anti-glioma pre-clinical investigations.Silencing GLS or overexpressing GLS2 induces growth inhibition in glioma cell lines. Inhibition is synergistically enhanced after arsenic trioxide (ATO) or H2O2 treatment. Glutatione levels decrease in GLS-silenced cells but augment if GLS2 is overexpressed. ROS synergistically inhibit cell migration by GLS silencing or GLS2 overexpression. c-myc, bid, and bcl-2 mediate apoptosis resulting from GLS silencing or GLS2 overexpression.

Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells, in order to study its effect on the hexosamine pathway and the pattern of protein O-glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose-6-P amidotransferase activity, which is the rate-limiting step of the hexosamine pathway. Although the overall cellular protein O-glycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase (OGT), the enzyme that links N-acetylglucosamine to proteins, was fivefold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins permitted the detection of two new OGT target proteins: the chaperonin TCP-1 theta and the oncogene Ets-related protein isoform 7. Taken together, our results support the hexosamine pathway and the O-glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell.

BackgroundGlutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.Methodology/principal findingsWe demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.Conclusions/significanceThis is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals.

Ehrlich ascites tumor cells (EATC) is a highly proliferative malignant cell line derived from mouse mammary epithelia, whereas their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, show a less transformed phenotype and loss of their tumorigenic capacity in vivo correlated with an inhibition of glutaminase expression. The mRNA differential display technique was applied to these two cell lines for the identification and isolation of genes whose transcription was altered. Side-by-side comparisons of cDNA patterns among relevant RNA samples revealed four genes significantly downregulated in 0.28AS-2 cells: high-mobility group Hmga2 protein, Fmnl3 or formin-like protein 3, Nedd-4 ubiquitin-protein ligase, and ubiquitin carboxyl-terminal hydrolase Usp-15. These positives were confirmed by Northern analysis. The four targeted genes have relevant functions in cell growth and proliferation. Our results show the validity of mRNA differential display technique to get insights into the molecular mechanisms underlying the acquisition of a more differentiated phenotype by tumor cells after inhibition of glutaminase expression.