FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF TUMOR SUPPRESSOR GENES IN STS

本提案的主要目标是继续 细胞突变和表达模式改变的表征 与肿瘤发生和肿瘤生长过程相关的周期调节剂 成人软组织肉瘤的进展。 是调查人员 假设TP 53和RB基因以及分子异常 那些直接或间接影响其编码产品的人， 肿瘤生长的选择性优势和软组织中的侵袭性行为 组织肉瘤患者。 这一点得到以下事实的支持： p53和pRB的突变和表达改变是常见的事件， 成人软组织肉瘤，并与不良的临床结果 降低了病人的存活率 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 其他计划成员在新的潜在肿瘤标志物的评价。 具体目的是：目的#1：进一步定义TP 53突变， 原发性和转移性软组织中p53和pRB表达模式的改变 组织肉瘤 研究人员计划前瞻性地验证以前的 他们的实验室和其他小组的报告显示， TP 53的预后价值。 他们还将确定是否有不同的TP 53 突变对疾病的结果有影响，如果有任何突变， 改变的p53和pRB表达模式之间的相关性。 这些研究 将与项目1和项目3以及 病理学和生物统计学核心。 目标二： 表征 p53突变产物和pRB改变的蛋白质的功能活性 在软组织肉瘤和STS细胞系中鉴定， 程序的组成部分。 p53和pRB的功能研究将是 进行了区分沉默突变从那些有助于 恶性表型 此外，调查人员还将研究- 流事件的途径，包括mdm 2和E2 F蛋白。 这些 研究将与Pavietich博士和Dr. Berrtino的实验室（见项目#3）。 目标#3：转移野生型 将TP 53转化为软组织肉瘤细胞进行TP 53突变处理，包括 STS细胞系在程序中建立。 研究人员建议 通过腺相关病毒载体将TP 53基因导入软组织肉瘤细胞 病毒载体和阳离子脂质体，试图恢复TP 53肿瘤 抑制功能。 他们还将分析特定的分子， 在TP 53野生型基因转移后经历差异表达， 包括mdm 2和E2 F1; cdk 2和cdk 4;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/WAF 1）。 调查人员将 还通过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).