Dual inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins occurs commonly during carcinogenesis. Inactivation of pRb leads to dysregulation of the E2F transcription factor family. E2F promotes both cell-cycle progression and apoptosis. The latter involves both p53- dependent and p53-independent mechanisms. My preliminary data suggests that E2F-1 can induce a recently identified p53 homolog called p73. p73 can, at least when overproduced, activate the transcription of p53-responsive genes and inhibit cell growth by inducing apoptosis. Since endogenous levels of p73 are very low and unlike p53, upstream signaling pathways leading to p73 activation have yet to be elucidated, it is intriguing that E2F-1 induces a significant increase in p73 levels. Dysregulation of the E2F/pRb pathway is common to most tumor cells. Therefore understanding the function of p73 within this pathway may provide clues as to the role of p73 in tumorigenesis. The goal of Specific Aim 1 is to determine the mechanism whereby E2F-1 induces p73. Standard protein stability and quantitative RNA assays will be employed to determine whether the effect of E2F-1 on p73 is due to changes in transcription, mRNA stability, and/or post-translational modifications. Whether the induction of p73 by E2F-1 in p53 nullizygous cells translates into activation of p53 target genes and apoptosis will form the basis of Specific Aims 2 and 3. The ability of E2F-1 to induce p73 dependent transactivation of p53 and p73 target genes will be assayed both biochemically and in transcription-based functional assays.
In Specific Aim 3, flow cytometry and immunofluorescence techniques will be used to investigate whether p73 contributes to E2F-1 dependent apoptosis. p73 dominant negative proteins will be used to determine specificity in these assays. Finally, in Specific Aim 4 the role of p73 in E2F- dependent apoptosis induced by chemotherapeutic agents will be investigated. These studies may provide the first clues as to the signals that regulate p73 and may also help to explain p53 independent killing by E2F. Ultimately, the results of the studies proposed in this application may help in the development of novel gene therapies in which the E2F-1 protein, or perhaps, p73, can be used to induce tumor cell apoptosis.
