N-MYC ONCOGENE REGULATION IN HUMAN NEUROBLASTOMA

人类神经母细胞瘤中 N-MYC 癌基因的调控

• 批准号: 2270114
• 负责人: RANDAL K WADA
• 金额: $ 9.67万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2270114
• 关键词: DNA binding protein; DNA footprinting; HeLa cells; cell differentiation; crosslink; gene deletion mutation; gene expression; gene mutation; genetic library; genetic promoter element; muscle tone; neoplasm /cancer classification /staging; neuroblastoma; nucleic acid probes; nucleic acid sequence; oncogenes; polymerase chain reaction; radiotracer; retinoid binding proteins; retinoids; transcription factor; transfection

The broad objectives of this proposal are to identify the DNA control elements responsible for regulating promoter activity of the N-myc oncogene in human neuroblastoma, and to determine whether derangements in this regulation have clinical significance. Preliminary studies have identified regions of the N-myc promoter that mediate its down regulation by retinoic acid (RA response element, or RARE), and its silencing in non-expressing, nonneuroblastoma cells. In addition these results have suggested that in some cases RARE mutation may be responsible for resistance to RA induced differentiation, and that lack of a silencer binding regulatory protein present in non-expressing cells may result in the constitutive basal N-myc expression in neuroblastoma. The first specific aim is to identify and characterize the RARE responsible for mediating the decrease in N-myc transcription observed upon RA induced differentiation of neuroblastoma cells. This will be accomplished by functional genetic analysis using transient transfections of neuroblastoma cell lines before and after RA treatment. In addition, biochemical analyses using gel retardation and footprinting will be performed to correlate function of these sequences with specific DNA- protein binding. Lastly the critical nucleotide positions within the RARE will be determined by observing the effect of specific mutations on RARE function and protein binding. The second specific aim is to identify the DNA sequence and proteins(s) mediating the silencing of the N-myc promoter in non-neuroblastoma cells. Using N-myc expressing neuroblastoma, and non-expressing, non- neuroblastoma cell lines, genetic and biochemical analyses employed in the study of the RARE will be applied to the cell type specific silencer to determine its critical sequence. Regulatory protein(s) binding to this sequence will be characterized, and cloned y screening of cDNA expression library with the binding sequence probe. The third specific aim is to determine whether deficiencies in these regulatory regions and/or DNA binding protein(s) are associated with resistance to RA induced differentiation, and correlate with adverse clinical outcome. This will be examined by PCR amplifying and sequencing RARE and cell type specific silencer elements in a panel of N-myc expressing, RA sensitive and resistant neuroblastoma cell lines, in non- expressing, non-neuroblastoma cell lines, as well as in stage IV N-myc expressing tumors (persistantly aggressive with poor patient outcome), N-myc expressing stage IVS tumors (initially metastatic, but undergo N- myc down regulation and differentiation to benign ganglioneuroma), non- expressing, differentiated ganglioneuromas, and normal tissue. As an initial indication of silencer binding protein production, reverse transcription PCR will be used to quantitate its mRNA expression in N-myc expressing and non-expressing cell lines, as well as in tumors and normal tissue. Such knowledge should aid in patient prognostication, provide insight into the use of differentiating agents in the treatment of neuroblastoma, and suggest new therapeutic approaches based on altering tumor cell behavior by modulating oncogenic expression.

该提案的主要目标是确定 DNA 控制 负责调节 N-myc 启动子活性的元件 人神经母细胞瘤中的癌基因，并确定是否紊乱 此调节具有临床意义。 初步研究有 确定了 N-myc 启动子介导其下调的区域 视黄酸（RA 反应元件，或 RARE），及其沉默 非表达、非神经母细胞瘤细胞。 此外，这些结果还有 表明在某些情况下，RARE 突变可能是造成 对 RA 诱导分化的抵抗力，以及缺乏沉默子 结合非表达细胞中存在的调节蛋白可能会导致 神经母细胞瘤中 N-myc 的组成型基础表达。 第一个具体目标是识别和表征稀有物质 负责介导观察到的 N-myc 转录减少 RA 诱导神经母细胞瘤细胞分化。 这将是 通过使用瞬时转染的功能遗传分析来完成 RA 治疗前后神经母细胞瘤细胞系的变化。 此外， 使用凝胶阻滞和足迹法的生化分析将 将这些序列的功能与特定的 DNA 关联起来 蛋白质结合。 最后是关键核苷酸位置 RARE将通过观察特定突变的影响来确定 RARE 功能和蛋白质结合。 第二个具体目标是鉴定 DNA 序列和蛋白质 介导非神经母细胞瘤细胞中 N-myc 启动子的沉默。 使用表达 N-myc 的神经母细胞瘤和不表达 N-myc 的神经母细胞瘤 神经母细胞瘤细胞系、遗传和生化分析用于 RARE的研究将应用于细胞类型特异性沉默器 以确定其关键顺序。 调节蛋白结合 该序列将被表征，并通过 cDNA 筛选进行克隆 具有结合序列探针的表达文库。 第三个具体目标是确定这些方面是否存在缺陷 调节区和/或 DNA 结合蛋白与 对 RA 诱导分化的抵抗力，并与不良反应相关 临床结果。 这将通过 PCR 扩增和测序进行检查 N-myc 组中的 RARE 和细胞类型特异性沉默元件 表达，RA 敏感和耐药的神经母细胞瘤细胞系，在非 表达非神经母细胞瘤细胞系，以及 IV 期 N-myc 表达肿瘤（持续侵袭，患者预后不佳）， 表达 N-myc 的 IVS 期肿瘤（最初是转移性的，但经过 N- myc 下调并分化为良性神经节神经瘤），非 表达、分化的神经节神经瘤和正常组织。 作为一个 沉默子结合蛋白产生的初步迹象，反向 转录PCR将用于定量其在N-myc中的mRNA表达 表达和非表达细胞系，以及肿瘤和正常细胞 组织。 这些知识应该有助于患者的预测，提供洞察力 使用分化剂治疗神经母细胞瘤， 并提出基于改变肿瘤细胞的新治疗方法 通过调节致癌表达来行为。