FUNCTIONAL ASPECTS OF THE RETINOBLASTOMA GENE

本提案的一个长期目标是了解 人视网膜母细胞瘤（Rb）基因及其在启动和 各种人类肿瘤的进展，包括那些作为第二种肿瘤发生的肿瘤， 遗传性视网膜母细胞瘤患者的恶性肿瘤。的作用 这些常见的癌症如乳腺癌和肺癌也将被研究， 以及其可能的功能向内生长调节，永生化， 分化 具体的目标包括确定放置正常的 13号染色体或Rb cDNA本身进入人肿瘤细胞类型， 两个Rb等位基因的丢失也是有牵连的。这些包括 视网膜母细胞瘤，骨肉瘤，纤维肉瘤，乳腺癌， 小细胞肺癌和膀胱癌。一个主要重点 将被放置在Rb基因产物水平上，使用高亲和力Rb 我们实验室生产的抗体。这方面的研究 将包括检查Rb蛋白的存在或不存在， 单细胞水平，与Rb基因的磷酸化模式相关 具有永生化、生长调节、分化或 肿瘤发生和表征两种Rb相关蛋白， 我们最近记录的情况。此外，该机制 负责非磷酸化Rb蛋白的显著减少， 同时增加的磷酸化Rb蛋白形式发现， 将研究自发转化的成纤维细胞， 人上皮来源的自发转化细胞也将被 检查类似的Rb蛋白变化。 这些研究将采用各种方法。其中包括基因 转染转移，微细胞融合染色体转移， 通过免疫沉淀进行蛋白质分析。蛋白质印迹法， 免疫组化和二维凝胶电泳及Rb分析 和Rb相关蛋白。 这些研究应该提供关于Rb作用的关键信息， 基因在许多人类癌症的发生和发展中也起作用 作为Rb基因磷酸化的特定变化的作用， 产品在永生化，生长调节和分化， 人体细胞

A long term objective of this proposal is to understand the function of the human retinoblastoma (Rb) gene and its role in the initiation and progression of various human tumors including those which occur as second malignancies in individuals with hereditary retinoblastoma. Its role in such common cancers such as breast and lung will also be investigated as well as its possible function ingrowth regulation, immortalization, and differentiation. Specific aims include determining the effect of placing either a normal 13 chromosome or the Rb cDNA itself into human tumor cell types in which the loss of both Rb alleles has been implicated. These include retinoblastoma, osteosarcoma, fibrosarcoma, carcinoma of the breast, small cell carcinoma of the lung, and bladder cancer. A major emphasis will be placed on the Rb gene product level using a high affinity Rb antibody which has been produced in our laboratory. Studies in this area will include examining the presence of absence of the Rb protein at the single cell level, correlating the phosphorylation pattern of the Rb gene product with immortalization, growth regulation, differentiation, or tumorigenesis and characterizing two Rb associated proteins whose presence we have recently documented. In addition the mechanism responsible for the marked diminution in unphosphorylated Rb protein and concomitant increase in phosphorylated Rb protein forms found in spontaneously transformed fibroblasts will be studied and other spontaneously transformed cells of human epithelial origin also will be examined for similar Rb protein changes. Various methodologies will be used for these studies. These include gene transfer by transfection, chromosome transfer by microcell fusion, protein analysis by immunoprecipitation. Western blotting, immunohistochemistry and 2-D gel electrophoresis and the analysis of Rb and Rb-associated protein by microsequencing. These studies should provide critical information on the role of the Rb gene in the initiation and progression of numerous human cancers as well as the role of specific changes in phosphorylation of the Rb gene products in immortalization, growth regulation and differentiation of human cells.