Rb/E2F in Hormone signaling of Prostate Epithelium

描述(由申请人提供)：该实验室收集了大量证据，支持视网膜母细胞瘤基因产物(PRB)及其转录调节伙伴E2F1在雄激素受体表达和功能调节中的新作用。这些发现对于调节前列腺上皮的生长和存活具有重要意义。小鼠胚胎中Rb基因敲除的致命性使基因中断实验无法检测Rb在前列腺癌中的这种作用。为了绕过这个问题并启动Rb在前列腺癌中的功能研究，我的实验室利用了一种创新的方法，在小鼠胚胎死亡之前从它们的Rb-/-前列腺癌前体雏形中拯救出来，用它我们已经产生了第一个活的Rb-/-前列腺组织和Rb-/-前列腺癌上皮细胞系。这些模型为研究Rb缺失对前列腺上皮的生理影响提供了一个独特的实验平台。利用这项技术，我们已经令人信服地观察到，RB的丢失导致未转化细胞AR蛋白和mRNA表达增加，并且在间质生长因子存在的情况下，AR活性增加。我们还发现，Rb的特异性丢失导致永生化的前列腺组织和细胞对凋亡刺激不敏感，可能是由于线粒体/caspase-9途径减弱所致。基于这些发现，我们假设Rb和AR是一种精确调控但知之甚少的机制的相互作用的组成部分，该机制控制前列腺上皮的生长和存活。这些研究将确定Rb/E2F1在体内培养的前列腺上皮细胞和重组前列腺组织中对AR转录和AR活性的调节作用。我们将确定Rb缺失是否导致对前列腺基质细胞分泌的旁分泌生长因子的AR反应性增加，并阐明以这种激素非依赖性方式调节AR功能的特定信号通路。最后，我们将确定凋亡的线粒体/caspase-9通路是否在RB-/-细胞和组织重组体中减弱。

DESCRIPTION (provided by applicant): This laboratory has compiled extensive evidence supporting a novel role for the retinoblastoma gene product (pRb) and it's transcriptional regulatory partner, E2F1 in the regulation of androgen receptor expression and function. These findings have important implications for the regulation of prostate epithelial growth and survival. The lethal nature of the Rb knockout in mouse embryos has precluded gene disruption experiments to examine such a role for Rb in the prostate gland. To circumvent this problem and to initiate functional studies of Rb in the prostate gland, my laboratory has utilized an innovative approach to "rescue" Rb-/- prostate precursor rudiments from mouse embryos prior to their death with which we have generated the first viable Rb-/- prostate tissues and Rb-/-prostate epithelial cell lines. These models provide a unique experimental platform with which to investigate the physiologic consequences of Rb deletion on prostate epithelium. Using this technology, we have made the compelling observation that the loss of Rb results in non-transformed cells that exhibit increased expression of AR protein and mRNA, and in the presence of stromal growth factors, increased AR activity. We have also discovered that the specific loss of Rb results in immortalized prostate tissue and cells that are insensitive to apoptotic stimuli possibly due to an attenuated mitochondrial/caspase-9 pathway. Based on these findings, we hypothesize that Rb and AR are interacting components of a precisely regulated but poorly understood mechanism that controls the growth and survival of prostate epithelium. The proposed studies will determine the role of Rb/E2F 1 in the regulation of AR transcription and AR activity in cultured prostate epithelial cells and in recombinant prostate tissue in vivo. We will determine if Rb deletion results in increased AR responsiveness to paracrine growth factors secreted by prostate stromal cells and elucidate the specific signaling pathways that regulate AR function in this hormone-independent fashion. Lastly we will determine if the apoptotic mitochondrial/caspase-9 pathway is attenuated in the Rb-/- cells and tissue recombinants.