Cancer is a dynamic disease that results from an imbalance between the deregulated expression of positive growth regulators (oncogenes) and the growth inhibitory effects of negative growth regulators (tumor suppressor genes). The inactivation of negative growth regulators such as p53 and Rb has been found in many tumor types, and in general occurs late in the neoplastic process. Functionally, p53 acts as a negative growth regulator by activating either a G1/S-phase cell-cycle checkpoint or by activating an apoptotic cell death pathway in response to DNA damaging agents or growth limiting conditions. Although it is well established p53's role in activation of a cell-cycle checkpoint in response to DNA damage is through transcriptional activation of downstream effector genes such as p21 and GADD 45, it does not seem that p53's role as a transcription factor is needed for activation of cell death by the same agents. Recent studies by several labs have demonstrated that the introduction of dominant oncogenes such as E1A and ras into embryonic stem cells that still possess functional wild-type p53 will poise them for apoptotic cell death when subsequently exposed to DNA damaging agents or growth restrictive stresses. If these cells possess mutated p53 or are devoid of wild-type p53 activity, they exhibit a significant reduction in their ability to undergo apoptosis when exposed to the same stresses. The predisposition of oncogenically transformed cells that possess wild-type p53 to apoptotic cell death coupled with the knowledge that many p53 mutations occur at the later stages of tumor formation suggests that cells possessing wild-type p53 are somehow selected against during tumor growth. We hypothesize that hypoxia or low oxygen conditions act as a tumor specific stress to select against oncogenically transformed cells that possess wild-type p53. Hypoxia, like ionizing radiation, causes the activation of wild-type p53 protein and induces growth arrest in established cell lines. However, exposure of minimally transformed cells to low oxygen conditions in the presence of serum and glucose induces apoptotic cell death that depends on the presence of wild-type p53. The goals of this study are to investigate the genetics of p53 mediated growth arrest and apoptosis induced by low oxygen conditions. We will examine the p53 dependence of hypoxia induced growth arrest and apoptosis in p53 genetically matched mouse, rat and human cells that are minimally transformed by the introduction of dominant oncogenes. By mixing different ratios of wild-type and mutant p53 cells, we will first attempt to demonstrate in cell culture and then in multicellular spheroids that hypoxia selects against the growth of oncogenically transformed cells that possess wild-type p53, and permits the survival and/or growth of mutant p53 cells. Finally, we will correlate the oxygen status of human head and neck tumors with their p53 genotype to investigate whether there is a correlation between these two parameters in human tumors. We feel that these experiments will provide mechanistic insight into how the tumor microenvironment can play a role in selecting for the clonal expansion of a tumor cell that has lost its ability to modulate cell proliferation.
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