FUNCTIONAL ASPECTS OF THE RETINOBLASTOMA GENE

这项建议的长期目标是了解 人视网膜母细胞瘤(Rb)基因及其在视网膜母细胞瘤发生和发展中的作用 各种人类肿瘤的进展，包括作为第二种发生的那些 遗传性视网膜母细胞瘤患者的恶性肿瘤。它在这方面的作用 像乳腺癌和肺癌这样的常见癌症也将被调查为 以及其可能的内生调节、永生化和 差异化。 具体目标包括确定将一个正常的 13号染色体或Rb基因本身转化为人类肿瘤细胞类型 这两个Rb等位基因的丢失也被认为是有关联的。这些措施包括 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 将被放置在使用高亲和力Rb的Rb基因产物水平上 我们实验室已经生产出的抗体。这一领域的研究 将包括检查是否存在Rb蛋白在 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 两种Rb相关蛋白在肿瘤发生中的作用 我们最近记录了它的存在。此外，该机制 对非磷酸化Rb蛋白的显著减少和 伴随而来的是磷酸化Rb蛋白形式的增加 自发转化的成纤维细胞将被研究和其他 人类上皮来源的自发转化细胞也将 检查是否有类似的Rb蛋白变化。 这些研究将使用不同的方法。其中包括吉恩 通过转基因转移，通过微细胞融合转移染色体， 免疫沉淀法进行蛋白质分析。Western blotting， 免疫组织化学、双向凝胶电泳法及Rb分析 和Rb相关蛋白进行微测序。 这些研究应该提供有关RB作用的关键信息 基因在多种人类癌症的发生和发展中的作用 作为Rb基因磷酸化的特异性变化的作用 永生化、生长调节与分化中的产品 人类细胞。

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.