Over the last 5 years, this laboratory has compiled extensive evidence indicating that growth and apoptotic signals in prostate epithelial cells converge at a common point regulated by the retinohlastoma gene (Rb). Due to the lethal nature of the Rb knockout, gene disruption experiments to confirm such a rote for Rb have never been accomplished. Our laboratory, in collaboration with Simon Hayward and Gerald Cunha, has utilized an innovative approach to """"""""rescue"""""""" Rb-/- prostate precursor rudiments from mouse embryos prior to their death to generate viable Rb-/- prostate tissue. This collaboration has resulted in a study demonstrating that Rb-/- prostate tissue recombinants were highly susceptible to hormonally induced prostate carcinogenesis (appendix F). This model has drawn much interest due the recapitulation of several key features of human prostate cancer, namely its progressive nature from dysplasia to carcinoma. My laboratory is currently trying to elucidate the molecular mechanism by which Rb loss predisposes prostate epithelium to hormonal carcinogenesis. To this end we have isolated Rb-/- prostate epithelial cell lines, termed PrERb-/-. Using these cell lines we have discovered that the specific loss of Rb results in immortalized prostate epithelium and tissue that is insensitive to apoptotic stimuli possibly due to an attenuated caspase-9 pathway. We have identified a putative Rb-regulated pathway in which c-myc and telomerase may promote immortalization of the Rb-/- phenotype. But the most interesting finding was the increased expression and DNA-binding activity of the androgen receptor (AR), suggesting that Rb and AR are interacting components coordinating physiologic signals that control the growth and survival of prostate epithelium. Based on these findings, we hypothesize that Rb, in its capacity to regulate cell cycle, cell survival and androgen responsiveness, is central to a precisely regulated but poorly understood mechanism that mediates the growth and survival of prostate epithelium, The proposed studies will determine if Rb deletion diminishes the cell's ability to growth arrest and senesce or undergo apoptosis in PrERb-/- prostate grafts in vivo and in the PrERb-/- cell line. We will determine if the mitochondrial/caspase-9 pathway is attenuated in the Rb-/- phenotype. We will examine the contribution of the c-myc/telomerase pathway to PrERb-/- immortalization in cultured cells and in vivo. Lastly, we will determine the role of Rb and E2F1 in the regulation of AR transcription and determine if PrERb-/- cells and grafts exhibit heightened androgen sensitivity and AR activity. ? ? ?
