MOLECULAR CHANGES DURING PROSTATE CARCINOMA PROGRESSION

前列腺上皮内瘤变(PIN)向侵袭性的转变 癌症的特征是两种主要调节蛋白的缺失， β4整合素及其配体层粘连蛋白5。 我们假设层粘连蛋白5丢失的机制是 转录后缺陷的存在。层粘连蛋白5的失效 组装会导致Beta4的不稳定，而Beta4因降解而丢失。这个 这些调节蛋白的丢失破坏了正常信号的稳定 促进前列腺癌进展的细胞途径。我们会 通过使用LNCaP细胞株来检验这一假设，该细胞株与 原发前列腺癌，表达三个层粘连蛋白亚链mRNAs 但未能组装出功能齐全的异三聚体层粘连蛋白5 蛋白。在目标1中，我们将研究三个层粘连蛋白5亚链mRNAs 通过对RT-PCR产生的cDNA产物片段进行测序来检测突变。 这些mRNAs还将接受体外翻译能力测试。 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， β4整合素的降解和细胞膜稳定性。我们会 同时研究纯化的层粘连蛋白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 然后是免疫沉淀，在信号转导的原因中， 抗酪氨酸、Sh2和Grb2特异性蛋白印迹 抗体。层粘连蛋白5在支持细胞生长中的功能作用， 黏附、迁移和致瘤性也将在目标3中进行研究。 这些研究将使用附着力的定量分析进行。 和迁徙建立在我们的实验室。在目标4中，我们将测试 α6和α3整合素在慢性粒细胞白血病中持续存在的临床意义 Beta4的缺席。

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.