One of the most important questions in cancer therapeutics is: what makes curable malignancies curable? Abundant evidence suggests that the answer resides largely within the genetic composition of the tumor cells. The recent explosion of interest in cell death has converged with this notion, suggesting that the response to stress (i.e. antineoplastics) may be profoundly regulated by tumor suppressor genes and oncogenes. P53, the most commonly mutated or deleted tumor suppressor in human cancer, is now recognized to play a double role which may be pivotal to cancer therapeutic success: activation of a cell cycle checkpoint in untransformed cells, and induction of apoptotic death in oncogene-transformed cells. In terms of survival consequences these two activities are diametrically opposed, and it is plausible that p53 function may regulate treatment efficacy by simultaneously protecting normal cells (which arrest and repair) while triggering death in tumor cells. This activity widens the therapeutic index in both directions, and abundant clinical data in humans has supported the concept that p53 status (best analyzed as p53 function) predicts treatment outcome in most if not all cancers. Animal models have played an important role in developing this concept. While considerable advances have been made in dissecting the mechanisms connecting p53 to cell cycle checkpoints, a major gap exists in our understanding of how p53 regulates the apoptosis program. That question represents the major focus of this grant application. To address this question we have used genetically defined combinations of rodent cell lines to generate a biochemically manipulatable cell-free system which activates the apoptosis program in a p53-dependent fashion. We have shown that p53 protein within the extracts plays an essential role in subsequent activation of caspases. Using this system we have been able to carry out biochemical fractionations which have identified both active intermediates (between p53 and caspases) as well as anti-apoptotic activities (within extracts of p53 deficient genetically matched tumor cells). We have established a series of readout extracts which permit the purification to homogeneity of factors within this pathway, and have so far fractionated one activating intermediate to a single 110 kd protein species. We have also partially purified (in progress) a specific inhibitory complex found in cells which are null for p53, as well as additional positive and negative regulators of the p53-apoptosis pathway. This proposal focuses on systematic efforts to exploit this versatile system to identify and characterize (both biochemically and within cells) these robust apoptosis regulating activities which may both determine treatment efficacy in curable cancers and identify impediments to successful outcome in resistant cancers.
