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. 2014 Mar;35(3):1855-62.
doi: 10.1007/s13277-013-1247-4. Epub 2013 Oct 6.

Silencing of GLS and overexpression of GLS2 genes cooperate in decreasing the proliferation and viability of glioblastoma cells

Monika Szeliga  1 , Małgorzata Bogacińska-Karaś, Aleksandra Różycka, Wojciech Hilgier, Javier Marquez, Jan Albrecht
Affiliations

Silencing of GLS and overexpression of GLS2 genes cooperate in decreasing the proliferation and viability of glioblastoma cells

Monika Szeliga et al. Tumour Biol. 2014 Mar.

Abstract

Glutamine (Gln) metabolism, initiated by its degradation by glutaminases (GA), is elevated in neoplastic cells and tissues. In malignant glia-derived tumors, GA isoforms, KGA and GAC, coded by the GLS gene, are overexpressed, whereas the GLS2-coded GAB and LGA isoforms, are hardly detectable in there. Our previous study revealed that transfection of T98G glioblastoma cells with GAB reduced cell proliferation and migration, by a yet unknown mechanism not related to Gln degradation. The question arose how simultaneous overexpression of GAB and inhibition of KGA would affect glioblastoma cell growth. Here, we used siRNA to silence the expression of Gls in T98G cells which were or were not stably transfected with GAB (TGAB cells). In both T98G and TGAB cell lines, silencing of Gls with siRNAs targeted at different sequences decreased cell viability and proliferation in a different, sequence-dependent degree, and the observed decreases were in either cell line highly correlated with increase of intracellular Gln (r > 0.9), a parameter manifesting decreased Gln degradation. The results show that combination of negative modulation of GA isoforms arising from GLS gene with the introduction of the GLS2 gene product, GAB, may in the future provide a useful means to curb glioblastoma growth in situ. At the same time, the results underscore the critical role of Gln degradation mediated by KGA in the manifestations of aggressive glial tumor phenotype.

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Figures

Fig. 1
Fig. 1
Silencing of GLS decreases the level of KGA and GAC transcripts. Relative expression of KGA (a, b) and GAC (c, d) transcripts measured vs beta-actin expression. The results are mean ± SD for five mRNA isolations from each cell line. *p < 0.01 vs untreated or treated with scrambled siRNA cells as tested with one-way ANOVA followed by Tukey’s test
Fig. 2
Fig. 2
Silencing of GLS decreases the level of KGA and GAC proteins. The graphs show quantification of KGA (a, b) or GAC (c, d) band intensity normalized to GAPDH. The results are mean ± SD for four protein isolations from each cell line. *p < 0.01 vs untreated or treated with scrambled siRNA cells as tested with one-way ANOVA followed by Tukey’s test. In the lowest part, representative Western blots show amounts of KGA, GAC, and GAPDH in protein extracts from T98G (e) or TGAB (f) cells treated with siGls3–7
Fig. 3
Fig. 3
Silencing of GLS increases intracellular level of Gln in T98G (a) and TGAB (b) cells. T98G and TGAB cells were transfected with siGls3–7. The level of intracellular Gln was measured at 48 h post-transfection. The results are mean ± SD for four independent determinations for each cell line. *p < 0.05 vs untreated cells and treated with scrambled siRNA (scr3); **p < 0.05 vs T98G counterparts as tested with one-way ANOVA followed by Tukey’s test
Fig. 4
Fig. 4
Silencing of GLS decreases survival of T98G (a) and TGAB (b) cells. T98G and TGAB cells were transfected with siGls3–7. Mitochondrial activity was assessed by the MTT test at 48 h post-transfection. The results are mean ± SD for four independent determinations for each cell line. *p < 0.05 vs untreated cells and treated with scrambled siRNA (scr3) as tested with one-way ANOVA followed by Tukey’s test
Fig. 5
Fig. 5
Silencing of GLS decreases proliferation of T98G (a) and TGAB (b) cells. T98G and TGAB cells were transfected with siGls3–7. Proliferation was assessed by the BrdU test at 48 h post-transfection. The results are mean ± SD for six independent determinations for each cell line. *p < 0.05 vs untreated cells and treated with scrambled siRNA (scr3) as tested with one-way ANOVA followed by Tukey’s test
Fig. 6
Fig. 6
Correlations between Gln level and cell viability or proliferation. a Correlation between Gln level and results of MTT assay in T98G cells (Pearson’s correlation coefficient: r = −0.8747, p = 0.01). b Correlation between Gln level and results of MTT assay in TGAB cells (r = −0.9937, p < 0.0001). c Correlation between Gln level and results of BrdU assay in T98G cells (r = −0.8925, p < 0.01). d Correlation between Gln level and results of BrdU assay in TGAB cells (r = −0.9774, p = 0.0001)
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