Szeliga Monika, Obara-Michlewska Marta, Matyja Ewa, Łazarczyk Marzena, Lobo Carolina, Hilgier Wojciech, Alonso Francisco J, Márquez Javier, Albrecht Jan
Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
Glia. 2009 Jul;57(9):1014-23. doi: 10.1002/glia.20825.
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.
肝型谷氨酰胺酶（LGA）是一种与转录调节有关的谷氨酰胺酶同工型。原发性胶质瘤术后样本中不存在LGA mRNA，且在培养的星形胶质细胞中含量较低。在本研究中，用携带人LGA序列的载体稳定转染T98G细胞，增加了LGA mRNA和蛋白的表达，以及细胞降解谷氨酰胺（Gln）的能力，表现为其稳态Gln含量降低三倍，谷氨酸（Glu）含量增加2.5倍。通过集落形成评估，转染细胞（TLGA细胞）的细胞存活率降低了40%，这与MTT试验所显示的线粒体活性显著降低密切相关。此外，与假转染细胞相比，细胞迁移减少了45%，增殖指数（Ki67免疫染色）降低了47%。包含超过47000个转录本的微阵列分析显示，TLGA中有85个基因的表达发生了显著改变，而在假转染细胞或对照细胞中则没有（P < 0.005）。通过实时PCR分析对八个可能与恶性肿瘤相关的基因（S100A16、CAPN2、FNDC3B、DYNC1LI1、TIMP4、MGMT、ADM和TIMP1）证实了微阵列数据。在这些变化中，S100A16和MGMT的表达降低与目前关于其蛋白产物在胶质瘤恶性肿瘤中作用的观点最为一致。胶质母细胞瘤细胞中新插入的LGA mRNA的恶性降低作用可以与这样一种假设相协调，即缺乏LGA mRNA的神经胶质来源肿瘤中不存在这种调节机制可能会促进其进展的某些方面。
