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Loss of p53 function through genetic mutations is selected for during tumor growth, at least in part because it allows tumor cell survival under the sub-optimal vascular supply and reduced oxygenation characteristic of growing solid tumors. Cells lacking an active p53 tumor suppressor display a diminished rate of apoptosis under hypoxic conditions. This is particularly relevant to glioblastoma, a malignant brain tumor showing abnormal vasculature with extensive areas of hypoxia and necrosis. Progression of lower grade astrocytoma to glioblastoma is marked with the clonal expansion of cells with mutant p53. The critical effectors of wt p53 function under hypoxic conditions have not yet been identified. To study the influence of reduced pO2 and p53 on tumorigenicity of glioblastoma we have monitored the changes in gene expression profiles using Affymetrix microarrays in a p53 null glioblastoma cell line (LN-Z308) containing tet-inducible p53 under normoxia and hypoxia. The list of p53-responsive genes identified contained many known p53 targets (confirming the accuracy of our experimental system) as well as novel p53-regulated genes. Target gene activation by p53 was confirmed by Northern blotting and their range of expression was examined in additional cellular models like the LN382 glioblastoma cell line with endogenous thermo-sensitive p53 mutant (having wild-type p53 conformation and function at 34C) or treatment of glioblastoma cell lines with genotoxic agents leading to the accumulation/activation of their endogenous wild-type p53 protein. We noticed that beside enzymes and transmembrane receptors, transcription factors appear to be the major group of genes regulated by p53 in glioblastoma, adding a layer of complexity to the p53-dependent network of gene expression. Interestingly, hypoxic conditions were found to strongly impact p53-regulated gene expression. Some target genes responded to p53 induction only in hypoxic conditions, while others like PDF/MIC-1 showed synergistic induction by both stimuli. To examine the importance of the genes discovered above for the tumorigenicity of glioblastoma we used an animal xenograft model. Re-expression of wild-type p53 by Tet-On system in p53-null glioblastoma completely abolished tumor growth in vivo. Next we allowed the p53-null tumor to reach the volume of 100-300 mm3 before re-expressing p53 and performed microarray analysis on ex vivo tumor specimens. These results are currently under analysis and will be compared to the results from the normoxic and hypoxic experiments in vitro.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]