Role of Retinoblastoma in Prostate Homeostasis

描述(申请人提供)：在过去的5年里，这个实验室收集了大量的证据表明，前列腺上皮细胞的生长和凋亡信号在一个公共点汇聚，受视网膜母细胞瘤基因(RB)调控。由于Rb基因敲除的致命性，证实Rb基因敲除的基因破坏实验从未完成过。我们的实验室与西蒙·海沃德和杰拉尔德·库尼亚合作，利用一种创新的方法，在小鼠胚胎死亡前将RB/前列腺癌前体雏形从它们的胚胎中拯救出来，以产生存活的RB/前列腺癌组织。这种合作导致了一项研究，表明RB-/-前列腺组织重组人对激素诱导的前列腺癌非常敏感(附录F)。由于重现了人类前列腺癌的几个关键特征，即从不典型增生到癌的进行性，该模型引起了人们的极大兴趣。我的实验室目前正试图阐明Rb缺失导致前列腺癌发生的分子机制。为此，我们分离了RB-/-前列腺上皮细胞系，命名为PrERb-/-。利用这些细胞系，我们发现Rb的特异性丢失导致永生化的前列腺上皮和组织对凋亡刺激不敏感，可能是由于caspase-9途径减弱所致。我们已经确定了一条假定的Rb调节途径，在该途径中，c-myc和端粒酶可能促进Rb-/-表型的永生化。但最有趣的发现是雄激素受体(AR)的表达和DNA结合活性增加，这表明Rb和AR是相互作用的组成部分，协调控制前列腺上皮生长和存活的生理信号。基于这些发现，我们假设Rb在调节细胞周期、细胞存活和雄激素反应的能力中处于核心地位，这是一种精确调控但知之甚少的调节前列腺上皮生长和存活的机制，拟议的研究将确定Rb缺失是否会降低体内和PrERb-/-前列腺癌移植物中细胞的生长停滞和衰老或经历凋亡的能力。我们将确定线粒体/caspase-9途径在Rb-/-表型中是否减弱。我们将研究c-myc/端粒酶通路在培养细胞和体内PrERb-/-永生化中的作用。最后，我们将确定Rb和E2F1在AR转录调控中的作用，并确定PrERb-/-细胞和移植物是否表现出更高的雄激素敏感性和AR活性。

DESCRIPTION (provided by applicant): 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.