FUNCTIONAL AND IMMUNOPHENOTYPIC ANALYSIS OF P53 AND RB

拟议研究的目的是继续表征突变和异常模式的细胞周期调节因子的表达，因为它们的利率在成人软组织肉瘤（STS）的肿瘤发生和肿瘤进展。我们的工作假设是TP 53和RB基因的突变和表达改变对STS患者的肿瘤生长和侵袭行为产生选择性优势。它们的关键作用需要其他因子如细胞周期蛋白、细胞周期蛋白依赖性激酶（Cdk）和细胞周期蛋白依赖性激酶抑制剂（Cki）的严格多水平调节。这些监管机构的研究是我们以前的工作的逻辑延伸。我们项目的主要目标是将基础和临床研究成果转化为临床研究。具体目标概述如下：目标#1。TP 53和RB在成人软组织肉瘤中的预后意义：分子和功能分析这一目标分为两项研究。在研究A中，我们计划：1）前瞻性验证TP 53和RB的预后价值; 2）确定不同TP 53突变的功能意义; 3）确定改变的pRB表达的分子基础以及E2 F水平与对化疗的响应的相关性。在研究B中，我们将确定特定STS亚型是否具有与p53和pRB相关的特定分子“特征”。目标2。p53通路的分子特征：对细胞周期阻滞和凋亡的影响。我们将确定：1）p21表达模式改变的临床相关性; 2）由于缺乏MDM 2扩增而过度表达mdm 2的STS的比例; 3）过度表达mdm 2但不表达p19/ARF的STS病例的比例;以及4）联合p53和mdm 2改变的潜在临床影响。目标3。RB途径的分子表征：对S期进入和增殖的影响。我们将确定：1）pRB过表达的频率、临床意义和性质; 2）STS中INK 4A的突变频率，以及这些突变是否涉及p16、p19/ARF或两者; 3）过表达细胞周期蛋白D1和Cdk 4的病例的比例，以及多大比例是由于基因扩增; 4）CDK 4和MDM 2共扩增的频率;和5）检测E2 F蛋白（主要是E2 F1、E2 F4和E2 F5）的改变的表达模式的频率和潜在的临床相关性。

The objective of the proposed studies is the continued characterization of mutations and aberrant patterns of expression of cell cycle regulators as they rate to tumorigenesis and tumor progression in adult soft tissue sarcomas (STS). Our working hypothesis has been that mutations and altered expression of the TP53 and RB genes produce a selective advantage for tumor growth and aggressive behavior in STS patients. Their critical role requires stringent multi-level regulation by other factors such as cyclins, cyclin-dependent kinases (Cdk), and cyclin-dependent kinase inhibitors (Cki). Studies of these regulators are a logical extension of our previous work. The main goal of our project is to translate basic and clinical research findings into clinical studies. The Specific Aims are outlined as follows: Aim #1. Prognostic Significance of TP53 and RB in Adult Soft Tissue Sarcomas: Molecular and Functional Analyses. This aim is divided into two studies. In Study A we plan to: 1) prospectively validate the prognostic value of TP53 and RB; 2) determine the functional significance of different TP53 mutations; 3) determine the molecular basis of altered pRB expression and relating E2F levels with response to chemotherapy. In Study B we will determine if particular STS subtypes have specific molecular "signatures" with respect to p53 and pRB. Aim #2. Molecular Characterization of the p53-Pathway: Impact on Cell Cycle Arrest and Apoptosis. We will determine: 1) the clinical relevance of altered patterns of p21 expression; 2) the proportion of STS that over- express mdm2 by lack MDM2 amplification; 3) the proportion of STS cases that over-express mdm2 but do not express p19/ARF; and 4) the potential clinical impact of joint p53 and mdm2 alterations. Aim #3. Molecular Characterization of the RB-Pathway: Impact on S- Phase Entry and Proliferation. We will determine: 1) the frequency, clinical significance, and nature of pRB over-expression; 2) the mutation frequency of INK4A in STS, and if these mutations involve p16, p19/ARF, or both; 3) the proportion of cases that over-express cyclin D1 and Cdk4, and what proportion is due to gene amplifications; 4) the frequency of co-amplification of CDK4 and MDM2; and 5 ) the frequency and potential clinical relevance of detecting altered expression patterns of E2F proteins (mainly E2F1, E2F4, and E2F5).