MOLECULAR CHANGES DURING PROSTATE CARCINOMA PROGRESSION

从前列腺上皮内瘤变（PIN）到浸润性前列腺增生的转变 癌的特征在于两种主要调节蛋白的缺失， β 4整联蛋白及其配体层粘连蛋白5。 我们假设层粘连蛋白5的丢失机制是： 存在转录后缺陷。层粘连蛋白5的失败 组装导致β 4的不稳定性，β 4由于降解而丢失。的 这些调节蛋白的丢失会破坏正常信号传导 前列腺肿瘤的发生与发展我们将 通过使用LNCaP细胞系来检验这一假设， 原发性前列腺癌，表达三种层粘连蛋白亚链mRNA 但不能组装功能完整的异源三聚体层粘连蛋白5 蛋白在目标1中，我们将检测三种层粘连蛋白5亚链mRNA， 通过对由RT-PCR产生的cDNA产物的片段进行测序来检测突变。 还将检测mRNA的体外翻译能力。 在目标2中，我们将测试纯化的层粘连蛋白5对合成的影响， 降解和β 4整联蛋白的细胞质膜稳定性。我们将 还研究了纯化的层粘连蛋白5引起信号转导的能力。 这些实验将利用脉冲标记或表面生物素化 随后是免疫沉淀，在信号转导的过程中， 蛋白质印迹法与抗磷酸酪氨酸，Sh 2，和GrB 2特异性 抗体的层粘连蛋白5对前列腺细胞生长的功能作用， 粘附、迁移和致瘤性也将在目标3中进行研究。 这些研究将使用粘附的定量测定进行 在我们的实验室里建立了迁移。在目标4中，我们将测试 整合素α 6和α 3持续存在的临床意义 没有Beta4。

The transition from prostatic intrepithelial neoplasia (PIN) to invasive carcinoma is characterized by a loss of two major regulatory proteins, beta4 integrin and its ligand, laminin 5. We hypothesize that the mechanism for the loss of laminin 5 is the presence of a post-transcriptional defect. The failure of laminin 5 assembly causes instability of beta4 which is lost due to degradation. The loss of these regulatory proteins destabilizes the normal signaling pathway of the cells contributing to prostate tumor progression. We will examine this hypothesis by using the LNCaP cell line which, like the primary prostate carcinomas, expresses the three laminin subchain mRNAs but fails to assemble the fully functioning heterotrimeric laminin 5 protein. In Aim 1 we will examine the three laminin 5 subchain mRNAs for mutations by sequencing fragments of the cDNA products produced by RT-PCR. The mRNAs will also be tested for their in vitro translational capacity. In Aim 2 we will test the effect the purified laminin 5 on the synthesis, degradation, and cytoplasmic membrane stability of beta4 integrin. We will also study the ability of purified laminin 5 to cause signal transduction. These experiments will utilize pulse labeling or surface biotinylation followed by immunoprecipitation, and in the cause of signal transduction, Western blotting with anti-phosphotyrosine, Sh2, and GrB2 specific antibodies. The functional effect of laminin 5 on prostrate cell growth, adhesion, migration, and tumorigenicity will also be studied in Aim 3. These studies will be carried out using quantitative assays of adhesion and migration established in our laboratory. In Aim 4 we will test the clinical relevance of the persistence of alpha 6 and alpha 3 integrins in the absence of beta4.