Mechanisms of Neoplastic Transformation by HMG-I/Y

描述(申请人提供)：HMG-I/Y基因编码HMG-I和-Y蛋白亚型，其功能是参与转录调控的结构染色质结合蛋白。这些蛋白在人类癌症中上调，尽管它们在恶性肿瘤发病机制中的作用尚不清楚。为了了解HMG-I/Y蛋白如何促进转化，我们正在探索它们的调控和功能。我们发现HMG-I/Y是Burkitt淋巴瘤中重要的c-Myc直接靶基因。我们还证明了HMG-I/Y是转化所必需的，因为减少人类癌细胞中这些蛋白的表达会阻止转化的表型。我们首次发现HMG-I/Y蛋白具有多种致癌特性。具体地说，在几个实验细胞系中，HMG-I或-Y的过度表达导致了不依赖于锚定的细胞生长。高表达HMG-I或-Y的成纤维细胞可在裸鼠体内致瘤。我们建立了在淋巴样细胞中过表达HMG-/的转基因小鼠，所有小鼠在平均8个月龄时都出现了淋巴样增生和恶性病变。HMG-I的过度表达也与基因组的不稳定性有关，这可能有助于肿瘤的发生或发展。因此，我推测HMG-I/Y是人类癌症发病机制中一个重要的癌基因。这项研究计划的重点是利用我实验室开发的独特试剂来确定HMG-I/Y转化的机制。我们的具体目标是：1.)利用微阵列技术鉴定与肿瘤转化相关的直接HMG-I/Y基因靶点。2.)确定HMG-I/Y参与转化、染色体不稳定和细胞周期调节的功能结构域。(A.)软琼脂转化法鉴定HMG-I/Y的功能域。B.)研究HMGI/Y在基因组不稳定性中的作用，并利用光谱核型分析确定相关结构域。(C)研究HMG-I/Y在细胞周期调控中的作用，并通过细胞周期谱分析确定相关结构域。3.)使用我们的HMG-I转基因小鼠确定转化过程中涉及的途径。(A.)评估HMG-I转基因小鼠转化的淋巴样细胞是否过表达HMG-I靶基因和染色体不稳定B。)通过将HMG-I转基因小鼠与其他基因改变的小鼠杂交，确定HMG-I转化所涉及的途径。4.)确定HMG-I/Y在淋巴系统恶性肿瘤和脑肿瘤中的表达是否与预后和临床预后相关。研究HMG-I/Y基因和蛋白在约翰·霍普金斯白血病和脑肿瘤库患者样本中的表达。这一建议意义重大，因为这一结果将增强我们对HMG-I/Y蛋白增加的人类恶性肿瘤的理解，并可能导致新的治疗策略。

DESCRIPTION (provided by applicant): The HMG-I/Ygene encodes the HMG-I and -Y protein isoforms, which function as architectural chromatin binding proteins involved in transcriptional regulation. These proteins are up-regulated in human cancer, although their role in the pathogenesis of malignancy is unclear. To understand how HMG-I/Y proteins may contribute to transformation, we are exploring their regulation and function. We discovered that HMG-I/Y is a direct c-Myc target gene important in Burkitt's lymphoma. We also demonstrated that HMG-I/Y is necessary for transformation because decreasing these proteins in human cancer cell lines blocks the transformed phenotype. We were the first to show that HMG-I/Y proteins have several oncogenic properties. Specifically, overexprossion of HMG-I or-Y leads to anchorage-independent cell growth in several experimental cell lines. Fibroblasts overexpressing HMG-I or-Y are tumorigenic in nude mice. We developed transgenic mice overexpressing HMG-/in lymphoid cells and all of them develop lymphoid hyperplasia and malignancy at a mean age of 8 months. HMG-I overexpression also correlates with genomic instability, which may contribute to tumor initiation or progression. Thus, I hypothesize that HMG-I/Y is an oncogene important in the pathogenesis of human cancer. The focus of this research proposal is to identify the mechanisms involved in transformation by HMG-I/Y using unique reagents developed in my laboratory. Our Specific Aims Are: 1.) Identify and characterize direct HMG-I/Y gene targets involved in neoplastic transformation using microarray analysis. 2.) Define the functional domains of HMG-I/Y involved in transformation, chromosomal instability, and cell cycle regulation. A.) Identify the functional domains of HMG-I/Y required for transformation using the soft agar transformation assay. B.) Investigate the role of HMGI/ Y in genomic instability and identify the relevant domains using spectral karyotyping analysis. C.) Investigate the role of HMG-I/Y in cell cycle regulation and identify the relevant domains using cell cycle profile analysis. 3.) Define the pathways involved in transformation using our HMG-I transgenic mice. A.) Assess transformed lymphoid cells from the HMG-I trangenic mice for overexpression of HMG-I target genes and chromosomal instability B.) Identify pathways involved in transformation by HMG-I by crossing the HMG-I transgenic mice with other genetically altered mice. 4.) Determine if HMG-I/Yexpression correlates with prognosis and clinical outcome in lymphoid malignancies and brain tumors. Study HMG-I/Y gene and protein expression in patient samples from the Johns Hopkins Leukemia and Brain Tumor Banks. This proposal is significant because the results will enhance our understanding of human malignancies with increased HMG-I/Y proteins and may lead to new treatment strategies.