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Transcription Factor Gata3 Expression Is Induced by Gls2 Overexpression in a Glioblastoma Cell Line but Is Gls2-Independent in Patient-Derived Glioblastoma

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Transcription Factor Gata3 Expression Is Induced by Gls2 Overexpression in a Glioblastoma Cell Line but Is Gls2-Independent in Patient-Derived Glioblastoma JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2017, 68, 2, 209-214 www.jpp.krakow.pl E. MAJEWSKA1, R. ROLA2, M. BARCZEWSKA3, J. MARQUEZ4, J. ALBRECHT1, M. SZELIGA1 TRANSCRIPTION FACTOR GATA3 EXPRESSION IS INDUCED BY GLS2 OVEREXPRESSION IN A GLIOBLASTOMA CELL LINE BUT IS GLS2-INDEPENDENT IN PATIENT-DERIVED GLIOBLASTOMA 1Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 2Department of Neurosurgery and Paediatric Neurosurgery, Medical University of Lublin, Lublin, Poland; 3Department of Neurosurgery, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland; 4Department of Molecular Biology and Biochemistry, Faculty of Sciences, Campus de Teatinos, University of Malaga, Malaga, Spain Phosphate-activated glutaminase (GA), a ubiquitous glutamine-metabolizing enzyme, is encoded by two genes, GLS and GLS2. In mammalian cancers, GLS isoforms are perceived as molecules promoting cell proliferation and invasion, whereas the role of GLS2 isoforms seems to be more complex and cell type-specific. Previous studies have shown abundance of GLS and lack of GLS2 transcripts in T98G human glioblastoma (GBM) cell line and patient-derived GBM. Transfection with GAB sequence, the whole GLS2 cDNA transcript, suppressed malignant phenotype of T98G cells. Microarray analysis revealed upregulation of GATA3, the product of which has been implicated in suppressing growth of some peripheral cancers. In this study we confirmed a significant upregulation of GATA3 expression in the transfected cells both at mRNA and protein level. Considerable expression of GATA3 was also observed in GBM tissues (previously shown as not expressing GLS2), while only traces or no GATA3 was detected in (GLS2-expressing) non-tumorigenic brain samples. In conclusion, while mechanistic relation between GAB and GATA3 expression is evident following in vitro manipulation of GBM cell line, it does not appear to be an intrinsic property of GBM nor non-tumorigenic brain tissue. Key words: glutaminase isoforms, GLS2 gene, GATA3, transcription factor, human glioblastoma cell line, glioblastoma tissue, non-tumorigenic brain tissue INTRODUCTION GBM cells (13, 14). Stable transfection of human glioblastoma cell line T98G with a sequence encoding GAB leads to Glioblastoma multiforme (GBM, WHO grade IV) belongs to suppression of malignant phenotype of these cells by decreasing the most malignant primary brain tumors with very poor prognosis their proliferation, viability and ability to migrate (15). (1). Glutamine (Gln) is a versatile amino acid promoting Moreover, the transfected cells, herein termed TGAB, are more hallmarks of malignancy (2). Phosphate-activated glutaminase sensitive to the alkylating agents used in glioma therapy (16). (GA, EC 3.5.1.2) metabolizes Gln to glutamate (Glu) and The exact mechanism of action of GAB remains unknown. Its ammonia and thus plays a significant role in tumor biology. nuclear localization in defined populations of neurons (17) and Human GA is encoded by two genes: GLS encodes kidney-type astrocytes (18) led to hypothesis that this molecule can modulate isoforms and GLS2 encodes liver type isoforms (3). So far, two gene expression. Consistent with this hypothesis, microarray transcripts arising from each of GA genes have been identified: analysis revealed that transfection of T98G cells with GAB KGA and GAC for GLS and GAB and LGA for GLS2 (3). sequence deregulated expression of 85 genes (15). Different studies revealed that glutaminase isoforms play GATA3 is one of the genes whose expression is upregulated opposite roles in tumorigenesis. GLS isoforms are associated in TGAB cells as compared to the controls (15). This gene codes with high rate of cell proliferation, whereas GLS2 proteins are for a multifunctional transcription factor which plays an related to resting or quiescent cell states (4). Suppression of the important role in the development of several tissues including malignant phenotype can be obtained by inhibition of GLS (5-7) brain (19). or overexpression of GLS2 (8, 9). Of note, the GLS gene has Available data on the role of GATA3 in the progression and been shown to be regulated by the mediators of oncogenesis, development of tumors of different tissue origin are ambiguous. MYC (10), Rho GTPases (6) and Notch (7), whereas the GLS2 The loss of GATA3 expression in breast cancer is linked to gene was identified as a target of p53 tumor suppressor (8). aggressive tumor development, metastatic capability and poor In GBM tissues GLS isoforms are highly expressed, while prognosis (20). The low level of GATA3 is related to the expression of GLS2 is silenced (11) via DNA hypermethylation promotion of cell migration and invasion of bladder cancer cells (12). Recent findings indicate that isoforms arising from GLS (21). Knockdown of GATA3 downregulated suppressors play opposing roles to isoforms encoded by GLS2 in growth of (UGT1A, PTEN, p53, p21) and upregulated oncogenes (i.e. c- 210 myc, cyclin D1, cyclin D3, cyclin E, FGFR3) in SVHUC tumorigenic brain tissues: two GBM adjacent tissues retrieved immortalized normal urothelial cells (22). In renal cell from the margin of the tumor and two other obtained by carcinoma GATA3 is silenced via hypermethylation (23). Low intracranial decompression for severe traumatic brain injury. The frequency of GATA3 expression has been detected in control tissues were identified by a neuropathologist as non- adenocarcinomas of lung, colon, endometrium, ovary and neoplastic. prostate (24). All the above would indicate a tumor suppressing role of GATA3. On the other hand, common GATA3 presence in Protein isolation and western blot analysis skin adnexal tumors, mesothelioma, salivary gland and pancreatic ductal carcinomas may not favor its tumor Cells or tissues were lysed in ice-cold RIPA buffer with suppressing role (24). Furthermore, overexpression of GATA3 cocktails of sodium fluoride, protease and phosphatase inhibitors correlates with a poor outcome of neuroblastoma patients (25). (Sigma-Aldrich). Lysates were centrifuged at 12,000 g for 10 Strong expression of GATA3 has also been documented in min at 4°C, and the supernatants were collected. Proteins Hodgkin lymphoma (HL) and down-regulation of GATA3 in HL concentrations were determined by bicinchoninic acid Protein cell lines demonstrated its role in the regulation of IL-5, IL-13, Assay Kit (Pierce, Rockford, IL, USA). Fifty µg of cells or tissue STAT4 (26). proteins were separated by 10% SDS-PAGE and then Previously performed microarray analysis revealed electrotransferred to nitrocellulose membranes. Membranes increased expression of GATA3 in TGAB cells as compared to were blocked with 5% skim milk for 1 hour at room temperature, the controls (15). This prompted the question whether the high and incubated with a human-specific anti-GATA3 antibody GATA3 content in GAB-enriched cells is a coincidence or (Abcam, Cambridge, MA) overnight at 4°C. After washing, reflects the regulation of expression of GATA3 by GAB. We first membranes were incubated with anti-rabbit antibody (Sigma- attempted to verify this hypothesis by comparing the mRNA and Aldrich) conjugated to horseradish peroxidase for 1 h at room protein level of GATA3 in T98G and TGAB cell lines. temperature. Blots were visualized on X-ray film using a Subsequently, we asked the question whether and in what degree SuperSignal West Pico Chemiluminiscence Substrate (Pierce). the finding obtained in a cell line is reflected in clinical For loading control, membranes were stripped for 15 min two glioblastomas which are composed of a heterogeneous times in Stripping Buffer and reused with an antibody against population of cells. To this end we measured the levels of glyceraldehyde 3-phosphate dehydrogenase (ProteinTech, GATA3 mRNA and protein in GLS2-negative GBMs tissues and Chicago, IL, USA). Densitometric analysis was performed with in non-tumorigenic brain tissues previously shown to be GLS2- G:Box system and GeneTools software (Syngene, Frederick, positive (12). MD, USA). Real-time PCR MATERIALS AND METHODS Total RNA from cell lines and tissues were extracted using a Cell lines and culture conditions commercially available TRI-Reagent (Sigma-Aldrich). Two µg of RNA were reverse-transcribed using High Capacity cDNA T98G human glioblastoma cell line (American Type Culture Reverse Transcription Kit (Applied Biosystems, Warrington, Collection), TGAB and TpcDNA cell lines were maintained in UK) according to the manufacturer’s protocol. The GATA3 Minimum Essential Medium Eagle (Sigma-Aldrich, St. Louis, primers were purchased from Applied Biosystems (assay ID: MO, USA) supplemented with 10% fetal bovine serum (Gibco, Hs00231122_m1), β-actin primers were purchased from Applied Thermo Fisher Scientific, Grand Island, NY, USA), 1% non- Biosystems (in case of cell line experiments, assay ID: essential amino acids (Gibco), and 1% antibiotics (penicillin and 4326315E) and Blirt (Gdansk, PL) (in case of biopsy material streptomycin) (Gibco). Cultures were maintained at 37°C in a experiments, cat no HK-DD-hu). Each reaction (total volume of humidified atmosphere with 5% CO2. TGAB and TpcDNA cell reaction 10 µl) contained 5 µl TaqMan Universal PCR Master lines were obtained by stable transfection of T98G cells with a Mix (Applied Biosystems), 1 µl cDNA, 0.5 µl primers and full cDNA sequence coding for human GAB or empty pcDNA3 RNAse/DNAse free water. The real time PCR reactions were vector respectively (15). The culture medium for TpcDNA
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