Investigating the role of BNIP3 and NIX in resistance of glioblastoma cells against hypoxia-induced apoptosis

Objectives: Glioblastoma multiforme (GBM) is among the most hypoxic tumors known and the oxygenation status of GBMs is negatively correlated with their therapy resistance, although the molecular mechanisms of this correlation remain largely unidentified. The aim of this study was to 1) analyze the variations of different glioblastoma cell lines in resistance to hypoxia-induced cell death, to 2) investigate the functional integrity of hypoxia-induced apoptotic signaling pathways in these cell lines and to 3) evaluate the relevance of alterations in apoptotic signaling for hypoxia resistance of GBM.

Methods: Transient expression assays in GBM cells were performed with expression plasmids encoding GFP-tagged BH3-only proteins BNIP3, NIX and Hrk. Cells were subjected to stringent hypoxia for 24, 48 or 72h in a hypoxic chamber (<0.1% oxygen, GasPak 150, Becton-Dickinson, Heidelberg, Germany). Hypoxia-induced cell death was analyzed by uptake of propidium iodide and subsequent FACS analysis (FACScalibur, Becton-Dickinson). Mitochondrial membrane potentials were measured by staining live cells with Tetramethylrhodamine methyl ester (TMRM) and subsequent FACS analysis (FACScalibur). RT-PCR, Western Blotting and immunofluorescence analysis was performed by standard procedures.

Results: Analysis of basal and hypoxia-induced BNIP3 and NIX expression in twenty glioblastoma cell lines revealed that loss of BNIP3 expression occurred in a significant number of the cell lines while it was potently induced by hypoxia in the remaining cell lines. In initial experiments, we established 48h of hypoxia as the optimal incubation time for studying hypoxia-induced cell death in GBM cells. Subsequent cell death quantification of a subset of the cell lines revealed drastic differences in sensitivity of individual cell lines to hypoxia-induced cell death. Quantification of cell death and analysis of mitochondrial dysfunction of all twenty cell lines is currently underway, but our preliminary data indicate a strong correlation between loss of BNIP3 expression and enhanced resistance to mitochondrial dysfunction and apoptosis in GBM cells.

Conclusions: Inactivation of hypoxia-induced apoptotic pathways might lead to enhanced resistance to mitochondrial dysfunction and apoptosis deficiency in GBM.

In particular, loss of expression of the putative tumor suppressor genes BNIP3 and NIX might be a common event in the genetic changes leading to GBM.