Experiments are described to understand mechanisms of tumor suppression by p53 and to characterize biochemical regulation of p53 and the cellular responses to p53 action. One series of experiments will use a well characterized in vivo model of multi-step carcinogenesis, Friend virus induced erythroleukemia, to study p53 as it becomes functionally engaged as a tumor suppressor. The virus causes a massive polyclonal expansion of red blood cell precursors, followed within weeks by the development of lethal erythroleukemia. Most tumors contain two significant mutations: (i) the activation of an Ets-related transcription factor, PU.1/Spil, by provirus insertion, and (ii) the functional loss of p53. The circumstances in which p53 loss is required for tumor progression will be determined by comparing tumor induction in wild type mice and mice that lack functional p53 genes. To test whether tumor suppressing functions of p53 are engaged by PU.1, cultured erythroblasts from wild type and p53 null mice will be infected with a PU.1-expressing retrovirus. PU.1 is expected to transform p53 null cells but to induce wild-type cells to undergo apoptosis at the time normal cells undergo terminal differentiation. The system will also be used to identify biochemical processes that may regulate p53 function and cellular responses to p53 action. In particular, levels of WAF1 transcripts increase during differentiation of wild type but not p53-null erythroblasts. The proposed experiments will determine if WAF1 expression results from activation of p53 expression or activity, or from other transcription factors that regulate activation of the WAF1 promoter by p53. Another series of experiments will use the U3NeoSV1 gene trap retrovirus to identify genes required for E1A/p53 dependent apoptosis. These studies will use cells that express adenovirus E1A and a temperature conditional p53 mutant, p53Val135, which undergo massive apoptosis upon transfer to 33oC. The strategy involves isolating libraries of Neo-resistant clones in which proviruses have disrupted most expressed genes and selecting for apoptosis resistant clones at 32oC. Apoptosis resistant clones will include those in which provirus integration has disrupted genes required for p53-dependent apoptosis. The shuttle vector, which allows disrupted genes to be cloned directly by plasmid rescue, will greatly simplify isolation of genes required for p53-dependent apoptosis. The proposed experiments are expected to provide fundamental information of the mechanisms of tumor suppression by p53. As p53 loss is associated with poor prognosis and with the resistance of tumors to therapeutic agents commonly used to treat cancer, these studies may suggest practical ways to reverse the consequences of p53 loss in the treatment human cancer.
| Peck, S R; Ruley, H E (2000) Ly108: a new member of the mouse CD2 family of cell surface proteins. Immunogenetics 52:63-72 |
| Kelley, L L; Hicks, G G; Hsieh, F F et al. (1998) Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage. Oncogene 17:1119-30 |
