Resistance to Hypoxia-Induced, BNIP3-Mediated Cell Death Contributes to an Increase in a CD133-Positive Cell Population in Human Glioblastomas In Vitro

ABSTRACTIn addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor–derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell–related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein–interacting protein 3)–dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5′-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5′-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.