Glutamine (Gln) plays a pivotal role in the metabolism of tumors of different including glioblastoma (GBM), the most aggressive brain tumor. Glutaminase (GA, EC 22.214.171.124) converts Gln to glutamate (Glu) and ammonia. GA is encoded by two genes: GLS and GLS2, encoding kidney-type isoforms (KGA and GAC) and liver-type isoforms (GAB and LGA), respectively. Kidney-type isoforms promote cell proliferation, while the liver-type isoforms relate to quiescent state of cells. In GBM GLS is highly expressed, while GLS2 is hardly detectable. Transfection of human GBM T98G cell line with a sequence encoding GAB is known to decrease their survival, proliferation index and migration and sensitizes them to damage by hydrogen peroxide. To examine whether the mode of action of GAB extends to other GBM cell lines, the effect of GAB transfection of U87MG, U251MG and LN229 cells with GAB was assessed.
Mitochondrial activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion (details in M. Szeliga et al., Glia, 2009). Cell proliferation was measured by a commercially available ELISA kit based on the detection of BrdU (5-bromo-2'-deoxyuridine) incorporated into the genomic DNA. Migration was analyzed using the scratch assay. The tip scratch of cell monolayer was photographed under Juli Smart cell analyzer and measured after 0 and 24 h. Ability to form colonies was assessed after 14 days of culture following Giemsa staining of fixed cells.
Transfection with GAB: i) decreased mitochondrial activity, proliferation and colony formation ability of U87MG cells ii) inhibited ability of U251MG cells to form colonies iii) decreased mitochondrial activity, proliferation, migration and colony formation ability of LN229 cells. All transfected cells were more sensitive to hydrogen peroxide as compared to the controls.
Suppression of malignant phenotype and their sensitization to hydrogen peroxide damage by GAB transfection appears to be a feature common to all the glioblastoma cell lines so far studied.
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Jan Albrecht, PhD
Ministry of Science and Higher Education of Poland, The Leading National Research Centre (KNOW-MMRC)
All authors have declared no conflicts of interest.