A close-up view of the Hunter syndrome.

The Lysosomal Storage disease known as Mucopolysaccharidosis type II, is caused by mutations affecting the iduronate-2-sulfatase required for heparan and dermatan sulfate catabolism. The central nervous system (CNS) is mostly and severely affected by the accumulation of both substrates. The complexity of the CNS damage observed in MPS II patients has been limitedly explored. The use of mass spectrometry (MS)-based proteomics tools to identify protein profiles may yield valuable information about the pathological mechanisms of Hunter syndrome. In this further study, we provide a new comparative proteomic analysis of MPS II models by using a pipeline consisting of the identification of native protein complexes positioned selectively by using a specific antibody, coupled with mass spectrometry analysis, allowing us to identify changes involving in a significant number of new biological functions, including a specific brain antioxidant response, a down-regulated autophagic, the suppression of sulfur catabolic process, a prominent liver immune response and the stimulation of phagocytosis among others.

Glutaminase isoforms expression switches microRNA levels and oxidative status in glioblastoma cells.

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.

Obesity and Stroke: Does the Paradox Apply for Stroke?

Historically, obesity has been identified as one of the most important risk factors for developing cardiovascular diseases including stroke; however, a theory called "The Obesity Paradox" has been recently considered. The paradoxical theory is that obese or overweight patients (according to body mass index score) can have better outcomes compared to leaner or malnourished patients. The paradox was initially discovered in patients with heart failure. The purpose of this manuscript was to investigate whether this paradox also applies to stroke patients, according to information available in the current literature.

Effect of glazing application side and mechanical cycling on the biaxial flexural strength and Weibull characteristics of a Y-TZP ceramic.

OBJECTIVE: Glaze application on monolithic zirconia (Y-TZP) can be a practical approach to improve the mechanical properties of this material. Our study evaluated the effect of glazing side and mechanical cycling on the biaxial flexure strength (BFS) of a Y-TZP. METHODOLOGY: Eighty sintered Y-TZP discs (Ø:12 mm; thickness: 1.2 mm - ISO 6872) were produced and randomly assigned into eight groups (n=10), according to the factors "glazing side" (control - no glazing; GT - glaze on tensile side; GC - glaze on compression side; GTC - glaze on both sides) and "mechanical aging" (non-aged and aged, A - mechanical cycling: 1.2×106, 84 N, 3 Hz, under water at 37°C). Specimens were subjected to BFS test (1 mm/min; 1,000 Kgf load cell) and fractured surfaces were analyzed by stereomicroscopy and SEM. Hsueh's rigorous solutions were used to estimate the stress at failure of glazed specimens. Two-way ANOVA, Tukey's test (5%), and Weibull analysis were performed. RESULTS: The "glazing side", "mechanical aging" and the interaction of the factors were significant (p<0.05). Groups GC (1157.9±146.9 MPa), GT (1156.1±195.3 MPa), GTC (986.0±187.4 MPa) and GTC-A (1131.9±128.9 MPa) presented higher BFS than control groups (Tukey, 5%). Hsueh's rigorous solutions showed that the maximum tensile stress was presented in the bottom of zirconia layer, at the zirconia/glaze interface. Weibull characteristic strength (σo) of the GC was higher than all groups (p<0.05), except to GT, GTC-A and GTC, which were similar among them. The fractography showed initiation of failures from zirconia the tensile side regardless of the side of glaze application and fatigue. CONCLUSION: Glazing zirconia applied on both tensile and compression sides improves the flexural strength of Y-TZP, regardless the mechanical aging.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Effect of glazing application side and mechanical cycling on the biaxial flexural strength and Weibull characteristics of a Y-TZP ceramic

Abstract Glaze application on monolithic zirconia (Y-TZP) can be a practical approach to improve the mechanical properties of this material. Objective Our study evaluated the effect of glazing side and mechanical cycling on the biaxial flexure strength (BFS) of a Y-TZP. Methodology Eighty sintered Y-TZP discs (Ø:12 mm; thickness: 1.2 mm - ISO 6872) were produced and randomly assigned into eight groups (n=10), according to the factors "glazing side" (control - no glazing; GT - glaze on tensile side; GC - glaze on compression side; GTC - glaze on both sides) and "mechanical aging" (non-aged and aged, A - mechanical cycling: 1.2×106, 84 N, 3 Hz, under water at 37°C). Specimens were subjected to BFS test (1 mm/min; 1,000 Kgf load cell) and fractured surfaces were analyzed by stereomicroscopy and SEM. Hsueh's rigorous solutions were used to estimate the stress at failure of glazed specimens. Two-way ANOVA, Tukey's test (5%), and Weibull analysis were performed. Results The "glazing side", "mechanical aging" and the interaction of the factors were significant (p<0.05). Groups GC (1157.9±146.9 MPa), GT (1156.1±195.3 MPa), GTC (986.0±187.4 MPa) and GTC-A (1131.9±128.9 MPa) presented higher BFS than control groups (Tukey, 5%). Hsueh's rigorous solutions showed that the maximum tensile stress was presented in the bottom of zirconia layer, at the zirconia/glaze interface. Weibull characteristic strength (σo) of the GC was higher than all groups (p<0.05), except to GT, GTC-A and GTC, which were similar among them. The fractography showed initiation of failures from zirconia the tensile side regardless of the side of glaze application and fatigue. Conclusion Glazing zirconia applied on both tensile and compression sides improves the flexural strength of Y-TZP, regardless the mechanical aging.

Bond strength between a polymer-infiltrated ceramic network and a composite for repair: effect of several ceramic surface treatments.

The effects of several ceramic surface treatments on bond strength of a polymer-infiltrated ceramic network and resin composite as repair material were evaluated. CAD-CAM blocks of a polymer-infiltrated ceramic network (Vita Enamic) were sliced and subjected to aging process, followed by embedding in acrylic resin. The bonding/repair area was treated as follows (n = 30): C- without treatment; UA- universal adhesive application; FM- 10% hydrofluoric acid and silane application; OM-airborne-particle abrasion with aluminum oxide and silane application; RP- tribochemical silica coating; and CA- surface grinding and application of universal adhesive. Composite resin cylinders were made on the treated surface. Specimens from each group were assigned randomly to two subgroups (n = 15) considering storage condition: Baseline (shear tests after 48 hours) or Storage (tests after 6 months under distilled water). The treated surfaces were analyzed by goniometry, roughness, and SEM. Two-way ANOVA and 1-way ANOVA were applied to analyze the bond data and roughness / contact angle data, respectively, followed by Tukey's test (α = 5%). Surface treatments and storage conditions affected bond strengths (p < 0.01). Surface grinding (CA) followed by universal adhesive promoted the highest value of bond strength (14.5 ± 4.8 MPa for baseline, 8.5 ± 3.4 MPa for storage) and the roughest ceramic surface. Grinding with silicon carbide paper (simulating diamond bur) followed by the application of a universal adhesive system is the best option for repairing fractures of the polymer-infiltrated ceramic network.

Bond strength between a polymer-infiltrated ceramic network and a composite for repair: effect of several ceramic surface treatments

Abstract: The effects of several ceramic surface treatments on bond strength of a polymer-infiltrated ceramic network and resin composite as repair material were evaluated. CAD-CAM blocks of a polymer-infiltrated ceramic network (Vita Enamic) were sliced and subjected to aging process, followed by embedding in acrylic resin. The bonding/repair area was treated as follows (n = 30): C- without treatment; UA- universal adhesive application; FM- 10% hydrofluoric acid and silane application; OM-airborne-particle abrasion with aluminum oxide and silane application; RP- tribochemical silica coating; and CA- surface grinding and application of universal adhesive. Composite resin cylinders were made on the treated surface. Specimens from each group were assigned randomly to two subgroups (n = 15) considering storage condition: Baseline (shear tests after 48 hours) or Storage (tests after 6 months under distilled water). The treated surfaces were analyzed by goniometry, roughness, and SEM. Two-way ANOVA and 1-way ANOVA were applied to analyze the bond data and roughness / contact angle data, respectively, followed by Tukey's test (α = 5%). Surface treatments and storage conditions affected bond strengths (p < 0.01). Surface grinding (CA) followed by universal adhesive promoted the highest value of bond strength (14.5 ± 4.8 MPa for baseline, 8.5 ± 3.4 MPa for storage) and the roughest ceramic surface. Grinding with silicon carbide paper (simulating diamond bur) followed by the application of a universal adhesive system is the best option for repairing fractures of the polymer-infiltrated ceramic network.

Glutaminases in brain: Multiple isoforms for many purposes.

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.

Both GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells.

UNLABELLED: 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. KEY MESSAGE: 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.

Mammalian glutaminase Gls2 gene encodes two functional alternative transcripts by a surrogate promoter usage mechanism.

BACKGROUND: Glutaminase 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 FINDINGS: We 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/SIGNIFICANCE: This 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.

O lugar do ambulatório na reformulação da assistência em saúde mental / The place of the clinic in the reformulation of mental health care

Este trabalho tem por objetivo discutir o lugar do ambulatório na atual Rede de Saúde Mental. Para isso, faz uma análise do processo de transformação do modelo de atenção presente no ambulatório tradicional para o modelo de atenção ambulatorial proposto pela Reforma Psiquiátrica. A partir da discussão de diversos autores e da experiência do ambulatório da cidade de Rio das Ostras, se propõe nesta pesquisa refletir os principais desafios e dificuldades enfrentadas pelos serviços ambulatoriais para a promoção de um cuidado integral diante da complexidade do campo da Saúde Mental.

Glutamine homeostasis and mitochondrial dynamics.

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.

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.

Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells.

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.

Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization.

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.

Identification of genes downregulated in tumor cells expressing antisense glutaminase mRNA by differential display.

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.

Sensitisation of Ehrlich ascitic tumour cells to methotrexate by inhibiting glutaminase.

BACKGROUND: Glutaminase activity is correlated with cancer proliferation and with growth rate in normal cells. Ehrlich ascites tumour cells (EATC) and their derivative 0.28AS-2 cells, which express antisense glutaminase mRNA, show differences in both morphology and tumorigenic capacity. MATERIALS AND METHODS: Cell viability was determined with the microtetrazolium cytotoxicity test assay. Immunofluorescence staining with annexin-V and propidium iodide was carried out to assess the number of apoptotic cells. RESULTS: 0.28AS-2 cells are less resistant to H2O2 than EATC, since half the concentration of H2O2 caused a similar effect on the cell population in 24 h. Methotrexate significantly inhibited the proliferation of both EATC and 0.28AS-2 cells at concentrations higher than 64 nM after 48 h of exposure. CONCLUSION: 0.28AS-2 cells are highly sensitised to methotrexate. These results provide insights into the possible role of glutaminase in cancer therapy by demonstrating that the expression of antisense mRNA for glutaminase decreases chemoresistance to some pro-apoptotic agents.

Inhibition of glutaminase expression increases Sp1 phosphorylation and Sp1/Sp3 transcriptional activity in Ehrlich tumor cells.

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.