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）及其转录调控伴侣E2 F1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生长和存活的调节具有重要意义。小鼠胚胎中Rb基因敲除的致命性已经排除了基因破坏实验来检查Rb在前列腺中的这种作用。为了解决这个问题，并启动Rb在前列腺的功能研究，我的实验室已经利用了一种创新的方法来“拯救”Rb-/-前列腺前体雏形小鼠胚胎死亡之前，我们已经产生了第一个可行的Rb-/-前列腺组织和Rb-/-前列腺上皮细胞系。这些模型提供了一个独特的实验平台，研究Rb缺失对前列腺上皮的生理后果。使用这种技术，我们已经取得了令人信服的观察结果，Rb的损失导致非转化细胞表现出增加的AR蛋白和mRNA的表达，并在基质生长因子的存在下，增加AR活性。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，这可能是由于线粒体/胱天蛋白酶-9途径减弱。基于这些发现，我们假设Rb和AR是一个精确调控但知之甚少的机制，控制前列腺上皮细胞的生长和存活的相互作用的组成部分。拟开展的研究将确定Rb/E2 F1在体外培养的前列腺上皮细胞和重组前列腺组织中调节AR转录和AR活性的作用。我们将确定Rb缺失是否会导致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，我们将确定凋亡线粒体/caspase-9途径是否在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.