Rb/E2F in Hormone signaling of Prostate Epithelium

描述（由申请人提供）：本实验室收集了大量证据，支持视网膜母细胞瘤基因产物（pRb）及其转录调控伙伴E2F1在雄激素受体表达和功能调控中的新作用。这些发现对前列腺上皮细胞生长和存活的调控具有重要意义。在小鼠胚胎中，Rb基因敲除的致死性阻碍了基因破坏实验来检验Rb在前列腺中的作用。为了规避这一问题并启动Rb在前列腺中的功能研究，我的实验室采用了一种创新的方法，在小鼠胚胎死亡之前从它们的胚胎中“拯救”Rb-/-前列腺前体雏形，由此我们产生了第一个有活力的Rb-/-前列腺组织和Rb-/-前列腺上皮细胞系。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，我们已经取得了令人信服的观察结果，Rb的缺失导致未转化的细胞表现出AR蛋白和mRNA的表达增加，并且在基质生长因子的存在下，AR活性增加。我们还发现Rb的特异性缺失导致永生化前列腺组织和细胞对凋亡刺激不敏感，这可能是由于线粒体/caspase-9通路减弱。基于这些发现，我们假设Rb和AR是一个精确调控但尚不清楚的机制的相互作用成分，该机制控制前列腺上皮的生长和存活。这些拟进行的研究将确定Rb/ e2f1在体外培养前列腺上皮细胞和重组前列腺组织中调控AR转录和AR活性的作用。我们将确定Rb缺失是否导致AR对前列腺基质细胞分泌的旁分泌生长因子的反应性增加，并阐明以这种激素不依赖的方式调节AR功能的特定信号通路。最后，我们将确定Rb-/-细胞和组织重组体中凋亡的线粒体/caspase-9通路是否减弱。

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.