Marta Obara-Michlewska

Glutamine in neoplastic cells: Focus on the expression and roles of glutaminases

Glutamine is an important source of energy for neoplastic tissues, and products of its metabolism include, among others, glutamate (Glu) and glutathione (GSH), the two molecules that play a key role in tumor proliferation, invasiveness and resistance to therapy. Glutamine hydrolysis in normal and transforming mammalian tissues alike, is carried out by different isoforms of glutaminases, of which the two major are liver-type glutaminase (LGA) and kidney-type glutaminase (KGA). This brief review summarizes available data on the expression profiles and activities of these isoenzymes in different neoplastic tissues as compared to the tissues of origin, and dwells on recent work demonstrating effects of manipulation of glutaminase expression on tumor growth. A comment is devoted to the emerging evidence that LGA, apart from degrading Gln for metabolic purposes, is involved in gene transcription; its enforced overexpression in glioma cells was found to reduce their proliferation and migration.

Transfection with liver-type glutaminase cDNA alters gene expression and reduces survival, migration and proliferation of T98G glioma cells.

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.

Down‐regulation of Kir4.1 in the cerebral cortex of rats with liver failure and in cultured astrocytes treated with glutamine: Implications for astrocytic dysfunction in hepatic encephalopathy

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na+‐K+‐2Cl− cotransporter‐1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide‐induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D‐[3H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT‐1 mRNA. Both Gln and ammonia decreased hypoosmolarity‐induced 86Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down‐regulation of Kir4.1 may mediate distinct aspects of Gln‐induced astrocytic dysfunction in HE.

Transfection of a human glioblastoma cell line with liver-type glutaminase (LGA) down-regulates the expression of DNA-repair gene MGMT and sensitizes the cells to alkylating agents

O6‐methylguanine‐DNA methyltransferase (MGMT) is a DNA‐repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth‐depressing transfection of a T98G glioma cell line with liver‐type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA‐transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non‐transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100–1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.

Synergistic action of hypoosmolarity and glutamine in inducing acute swelling of retinal glial (Müller) cells

High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration‐dependent, with half‐maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative‐nitrosative stress, and (iv) inhibitors of phospholipase A2 and cyclooxygenase. Glutamine‐induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A1 receptors. The data suggest that hypoosmolarity accelerates the swelling‐inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative‐nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction.

Astroglial NMDA receptors inhibit expression of Kir4.1 channels in glutamate-overexposed astrocytes in vitro and in the brain of rats with acute liver failure.

Astroglial inward rectifying Kir4.1 potassium channels are fundamental for the maintenance of ion and water homeostasis in the central nervous system (CNS). Down-regulation of Kir4.1 expression is observed in CNS disorders associated with excessive extracellular glutamate (Glu) accumulation, including hepatic encephalopathy related to acute liver failure (ALF). Here we demonstrate that prolonged (3 days) treatment of cultured rat cortical astrocytes with 2 mM Glu or 100 µM NMDA decreases the expression of Kir4.1 mRNA and protein. Inhibition by Glu of Kir4.1 mRNA expression was reversed by NMDA receptor antagonists MK-801 and AP-5 (each at 50 µM), and by a non-transportable inhibitor of Glu uptake TBOA (100 µM). MK-801 reversed the inhibitory effect of Glu on Kir4.1 protein expression. In contrast, transcription of Kir4.1 channels was not affected by: (i) a transportable Glu uptake inhibitor PDC (100 µM); (ii) by group I mGluR antagonist MTEP (100 µM); (iii) by antagonists of oxidative-nitrosative stress (ONS) in astrocytes, including the neuroprotective amino acid taurine (Tau; 10 mM), the NADPH oxidase inhibitor apocyanine (APO; 300 µM), the nitric oxide synthase inhibitor, L-NNA (100 µM), and a membrane permeable glutathione precursor, glutathione-diethyl ester (GEE; 3 mM). Down-regulation of Kir4.1 transcription in rats with ALF was attenuated by intraperitoneal administration of a competitive NMDA receptor antagonist memantine, but not by histidine, which reverses ONS associated with ALF. Collectively, the results indicate that over-activation of astroglial NMDA receptors, aided by as yet undefined effects of Glu entry to astrocytes, is a primary cause of the reduction of Kir4.1 expression in CNS disorders associated with increased exposure to Glu.

Gain of function of Kir4.1 channel increases cell resistance to changes of potassium fluxes and cell volume evoked by ammonia and hypoosmotic stress

The Kir4.1 channel is an inward rectifying potassium channel involved in the control of potassium and water movement in mammalian cells. To evaluate independently the role of Kir4.1 alone and without interaction with other cellular effectors, we compared (86)Rb fluxes and cell volume in Kir4.1 transfected cells (Kir4.1(+)) with cells transfected with an empty vector (Kir4.1(-)). Transfection with Kir4.1 neither increased (86)Rb uptake nor (86)Rb efflux from cells in isotonic medium. Pretreatment with ammonia (5 mM ammonium chloride) in isotonic medium produced a pronounced increase of (86)Rb uptake and a moderate decrease of cell volume in Kir4.1(-) but not in Kir4.1(+) cells. However, pretreatment evoked no change in (86)Rb efflux in either cell type. Hypotonic treatment (HT) markedly increased (86)Rb efflux in Kir4.1(-) cells and increased cell volume in both cell types. Although pretreatment with ammonia did not alter the effect of HT on (86)Rb efflux in either Kir4.1(+) or Kir4.1(-) cells, it potentiated the effect of hypotonic treatment in increasing cell volume in Kir4.1(-) cells. The results demonstrate that the presence of Kir4.1 in cells increases their resistance to alterations of potassium fluxes and/or cell volume imposed by ammonia and hypotonicity.

Modulation by kynurenine of extracellular kynurenate and glutamate in cerebral cortex of rats with acute liver failure

BACKGROUND Kynurenic acid (KYNA) modulates the glutamatergic tone by controlling neuronal glutamate (GLU) release. The present study tested the potential of the KYNA precursor, kynurenine (KYN) to counter increased extracellular GLU associated with the pathogenesis of hepatic encephalopathy accompanying acute liver failure (ALF). METHODS ALF was induced in adult rats by administration of a hepatotoxin, thioacetamide. KYNA and GLU were measured in the cerebral cortical microdialysates of control (saline-treated) and ALF rats using HPLC. The expression of mRNA coding for kynurenine aminotransferase II (KAT-II), the astrocytic enzyme converting KYN to KYNA, was assayed by real-time PCR. RESULTS Cerebral cortical extracellular KYNA was increased in ALF rats not treated with KYN, consistent with a previously observed increase of cerebral cortical KATII activity in this ALF model. Single intraperitoneal administration of KYN (50 mg/kg, 120 min before microdialysate collection), produced a further substantial increase of extracellular KYNA, paralleled by a decrease of extracellular GLU. In cultured cerebral cortical astrocytes, the cells which in situ are the primary target of blood-derived ammonia and other toxins liberated due to ALF, elevation of KAT-II mRNA expression was noted upon their incubation with KYN and the KYN precursor, tryptophan (Trp), which is normally elevated by ALF. CONCLUSIONS Administration of exogenous KYN to stimulate KYNA synthesis may help correcting excessive extracellular accumulation of GLU in cerebral cortex caused by ALF. The therapeutic potential of KYN in ALF appears to be fostered by increased expression of KAT-II in astrocytes upon exposure to KYN or Trp.

Glutaminases in slowly proliferating gastroenteropancreatic neuroendocrine neoplasms/tumors (GEP-NETs): Selective overexpression of mRNA coding for the KGA isoform.

Glutamine (Gln) is a crucial metabolite in cancer cells of different origin, and the expression and activity of different isoforms of the Gln-degrading enzyme, glutaminase (GA), have variable implications for tumor growth and metabolism. Human glutaminases are encoded by two genes: the GLS gene encodes the kidney-type glutaminases, KGA and GAC, while the GLS2 gene encodes the liver-type glutaminases, GAB and LGA. Recent studies suggest that the GAC isoform and thus high GAC/KGA ratio, are characteristic of highly proliferating tumors, while GLS2 proteins have an inhibitory effect on tumor growth. Here we analyzed the expression levels of distinct GA transcripts in 7 gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with low proliferation index and 7 non-neoplastic tissues. GEP-NETs overexpressed KGA, while GAC, which was the most abundant isoform, was not different from control. The expression of the GLS2 gene showed tendency towards elevation in GEP-NETs compared to control. Collectively, the expression pattern of GA isoforms conforms to the low proliferative capacity of GEP-NETs encompassed in this study.

Interstitial ion homeostasis and acid-base balance are maintained in oedematous brain of mice with acute toxic liver failure

ABSTRACT Acute toxic liver failure (ATLF) rapidly leads to brain oedema and neurological decline. We evaluated the ability of ATLF‐affected brain to control the ionic composition and acid‐base balance of the interstitial fluid. ATLF was induced in 10–12 weeks old male C57Bl mice by single intraperitoneal (i.p.) injection of 100 &mgr;g/g azoxymethane (AOM). Analyses were carried out in cerebral cortex of precomatous mice 20–24 h after AOM administration. Brain fluid status was evaluated by measuring apparent diffusion coefficient [ADC] using NMR spectroscopy, Evans Blue extravasation, and accumulation of an intracisternally‐injected fluorescent tracer. Extracellular pH ([pH]e) and ([K+]e) were measured in situ with ion‐sensitive microelectrodes. Cerebral cortical microdialysates were subjected to photometric analysis of extracellular potassium ([K+]e), sodium ([Na+]e) and luminometric assay of extracellular lactate ([Lac]e). Potassium transport in cerebral cortical slices was measured ex vivo as 86Rb uptake. Cerebral cortex of AOM‐treated mice presented decreased ADC supporting the view that ATLF‐induced brain oedema is primarily cytotoxic in nature. In addition, increased Evans blue extravasation indicated blood brain barrier leakage, and increased fluorescent tracer accumulation suggested impaired interstitial fluid passage. However, [K+]e, [Na+]e, [Lac]e, [pH]e and potassium transport in brain of AOM‐treated mice was not different from control mice. We conclude that in spite of cytotoxic oedema and deregulated interstitial fluid passage, brain of mice with ATLF retains the ability to maintain interstitial ion homeostasis and acid‐base balance. Tentatively, uncompromised brain ion homeostasis and acid‐base balance may contribute to the relatively frequent brain function recovery and spontaneous survival rate in human patients with ATLF. HighlightsAcute toxic liver failure (ATLF) causes cytotoxic oedema and blood extravasation in mouse brain.ATLF compromises brain interstitial fluid movement suggesting glymphatic system dysfunction.Brain extracellular K+, Na+, lactate and pH are not affected by ATLF.ATLF‐affected brain cells retain the ability to control interstitial ion homeostasis. Abbreviation, ADC; Apparent diffusion coefficient; ALF; Acute liver failure; AOM; Azoxymethane; ATLF; Acute toxic liver failure; BBB; Blood brain barrier; CSF; Cerebrospinal fluid; ISF; Interstitial fluid; EB; Evans blue; MRI; Magnetic resonance imaging; [K+]e; Extracellular potassium concentration; [Na+]e; Extracellular sodium concentration; [Lac]e; Extracellular lactate concentration