FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF TUMOR SUPPRESSOR GENES IN STS

这项提案的主要目标是继续 细胞突变和表达模式改变的特征 与肿瘤发生和肿瘤过程相关的周期调节因子 成人软组织肉瘤的进展。是调查人员 假设TP53和Rb基因的分子异常，以及 那些直接或间接影响其编码产品的产品，会产生 软组织中肿瘤生长的选择性优势和侵袭性行为 组织肉瘤患者。这得到了以下事实的支持：TP53 P；53和pRb的突变和表达改变是乳腺癌的常见事件 成人软组织肉瘤与不良临床结局相关 并降低了患者的存活率。这个项目的目标是翻译 将基础研究成果转化为临床研究，并与 其他计划成员参与评估新的潜在肿瘤标记物。 具体目标是；目标1：进一步定义TP53突变和 原发和转移性软组织中P53和pRb表达的变化 组织肉瘤。调查人员计划前瞻性地验证之前的 来自他们的实验室和其他组织的报告显示 TP53对预后的预测价值。他们还将确定不同的TP53 突变对疾病的结局有影响，如果有的话， P53和pRb表达模式改变的相关性。这些研究 将与项目1和3以及与 病理学和生物统计学核心。目标2：描述 P53突变产物和pRb改变蛋白的功能活性 在软组织肉瘤和STS细胞系中鉴定为 程序的组件。P53和pRb的功能研究将被 以区分沉默突变和那些导致 恶性表型。此外，调查人员还将向下研究- 其通路的流事件，包括MDM2和E2F蛋白。这些 研究将与Pavietich博士和Dr Pavietich博士合作进行。 Berrtino的实验室(见项目3)。目标3：转移野生型 将TP53导入软组织肉瘤细胞处理TP53突变，包括 在计划中建立的STS细胞系。调查人员提议 用腺相关基因导入软组织肉瘤细胞中的TP53基因 病毒载体和阳离子脂质体修复TP53肿瘤的实验研究 抑制功能。他们还将分析特定分子，这些分子可能 在TP53野生型基因转移中进行差异表达， 包括MDM2和E2F1；CDK2和CDK4；细胞周期蛋白D1、A和E；以及某些 细胞周期蛋白依赖性激酶抑制剂(即p21/WAF1)。调查人员将 并通过DNA断裂分析(TUNEL法)评估细胞凋亡。

Th main objective of this proposal focuses on the continued characterization of mutations and altered patterns of expression of cell cycle regulators as they relate to processes of tumorigenesis and tumor progression in adult soft tissue sarcomas. It is the investigators hypothesis that molecular abnormalities of TP53 and RB genes, as well as those that directly or indirectly affect their encoded products, produce a selective advantage for tumor growth and an aggressive behavior in soft tissue sarcoma patients. This is supported by the facts that TP53 mutations and altered expression of p;53 and pRB are frequent events in adult soft tissue sarcomas and are associated with poor clinical outcome and reduced patient survival. The goals of this project are to translate basic research findings into clinical studies, and to collaborate with other Program members in the evaluation of novel potential tumor markers. The Specific Aims are; Aim #1: To further define TP53 mutations and altered patterns of p53 and pRB expression in primary and metastatic soft tissue sarcomas. The investigators plan to prospectively validate previous reports from their laboratory and other groups that have shown the prognostic value of TP53. They will also determine if different TP53 mutations have an impact on the outcome of the disease, and if there is any correlation between altered p53 and pRB expression patterns. These studies will be performed in collaboration with Projects 1 and 3, as well as with pathology and Biostatistics Cores. Aim #2: To characterize the functional activities of the p53 mutant products and pRB altered proteins identified in soft tissue sarcomas and STS cell lines established as a component of the program. Functional studies of p53 and pRB will be conducted to distinguish silent mutations from those contributing to the malignant phenotype. In addition, the investigators will also study down- stream events of their pathways, including mdm2 and E2F proteins. These studies will be conducted in collaborative with Dr. Pavietich and with Dr. Berrtino's laboratory (see Project #3). Aim #3: To transfer wild-type TP53 into soft tissue sarcoma cells processing TP53 mutations, including STS cell lines established in the Program. The investigators propose to introduce the TP53 gene in soft tissue sarcoma cells by an adeno-associated viral vector and by cationic liposomes in an attempt to restore TP53 tumor suppression functions. They will also analyze specific molecules that may undergo differential expression upon TP53 wild-type gene transfer, including mdm2 and E2F1; cdk2 and cdk4; cyclins D1, A and E; and certain cyclin dependent kinase inhibitors (ie, p21/WAF1). The investigators will also assess apoptosis by the analysis of DNA breaks (TUNEL method).