Carolina Lobo

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.

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 θ 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. J. Cell. Biochem. 103: 800–811, 2008.

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.

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.

Ehrlich ascites tumor cells expressing anti-sense glutaminase mRNA lose their capacity to evade the mouse immune system

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‐α‐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.

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

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 messageSilencing 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.

A new approach to the synthesis of 4,5-dioxoaporphine alkaloids from preformed biaryl bond precursors

Abstract The synthesis of cepharadione-B and 2-demethoxy analogues is described. Starting from fluorenones, ring C was formed by cyclization of (biphenyl-2-yl)acetyl morpholines under Bischler-Napieralsky conditions. The photochemistry of chloroacetamides was used to form ring B. The cytotoxicity of these compounds on several tumor cell lines was evaluated.

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.

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.

Energy Diagrams for Enzyme-Catalyzed Reactions: Concepts and Misconcepts.

Despite the utility that energy diagrams have as a teaching and learning tool, a survey of their use, in seven popular Biochemistry textbooks, reveals that there is certain confusion around this topic. In our opinion, this confusion arises from the reluctance of authors to consider and indicate the conditions under which the reaction being represented occurs. For an enzyme‐catalyzed reaction, it should be stressed that, under conditions where the overall reaction is spontaneous, each elementary step must exhibit a negative free energy change, and this must be properly reflected in the progression profile of the reaction.