In vivo, tumor aggressiveness correlates with increased resistance to apoptosis. Additionally, anti-neoplastic therapies, including gamma-radiation and chemotherapeutic drugs, eliminate malignant cells by the induction of apoptosis. Therefore, it is to the advantage of tumor cells to overexpress cellular genes which protect against this process. bcl-2, an important modulator of apoptosis, is negatively regulated at the transcriptional and post-transcriptional level by tumor suppressor p53 and positively by activated Ras. In contrast, Bcl-2 is able to overcome p53-dependent apoptosis, perhaps by cooperating with myc to subcellularly localize p53 to the cytoplasm. Overexpression of bcl-2 is transforming when accompanied by either c-myc or activated ras. In colorectal and prostate cancers, greater than 90 percent of metastatic tumors overexpress the Bcl-2 protein, supporting a role for Bcl-2 in oncogenesis. Another tumor suppressor, WT1, has been demonstrated to modulate the bcl-2 promoter and to inhibit p53-mediated apoptosis. Preliminary data presented in this research proposal demonstrates that activated ras and three Ras-responsive transcription factors (AP-1, C/EBPbeta, and EGR-1) lead to the transactivation of the bcl-2 promoter, and that at least one of these transcription factors, C/EBPbeta, directly interacts with the promoter. Furthermore, tumor suppressors p53 and WT1 were able to inhibit positive transactivation of the bcl-2 reporter. Importantly, a high affinity bindking site for WT1 was identified in the bcl-2 promoter. The primary goals of this research proposal are to determine the functional consequence of oncogenic Ras and tumor suppressors, WT1 and p53, on bcl-2 expression and the significance of this dysregulation on oncogenic transformation.
Aim 1 will employ transient transfections, using bcl-2 reporters, to confirm the location of the AP-1, C/EBPbeta and EGR-1 responsive sites and to determine the importance of these sites for p53-dependent repression. M1p53 135 cells, which contain a temperature-sensitive p53 mutant, and cells isolated from p53-deficient mice will be employed to determine whether gamma-radiation and interleukin-6, physiological inducers of AP-1, Egr-1, and C/EBPbeta, protect cells against apoptosis by inducing Bcl-2 expression.
Aim 2 will determine whether oncogenic Ras elevates endogenous bcl-2 gene expression and to determine the importance of C/EBP and p53 on bcl-2 expression and on cellular transformation.
Aim 3 will determine whether the WT1 binding site(s) is required for p53-mediated repression of the bcl-2 promoter and to determine the functional consequence of WT1 expression on endogenous bcl-2 expression in vivo. Primary sporadic Wilms' tumors will be analyzed for elevated bcl-2 expression, and a WT1 mouse xenograft model will be characterized to determine whether the anti-apoptotic activity of Bcl-2 is critical for sustained growth of Wilms' tumor. These studies have important implications not only toward understanding the functional consequence of oncogenic Ras and tumor suppressors on bcl-2 modulation, but may provide important therapeutic insight as to how to eradicate solid tumors display overexpressed Bcl-2.
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