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: 3478745
• 负责人: RANDAL K WADA
• 金额: $ 9.31万
• 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/3478745
• 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 癌基因在人类神经母细胞瘤，并确定是否紊乱 在此调节中具有临床意义。 初步研究已经 鉴定了N-myc启动子中介导其下调的区域 视黄酸（RA反应元件，或RARE），及其沉默， 不表达，非神经母细胞瘤细胞。 此外，这些结果还 这表明，在某些情况下，RARE突变可能是负责 对RA诱导分化抗性，以及沉默子的缺乏 存在于非表达细胞中的结合调节蛋白可导致 神经母细胞瘤中N-myc的组成性基础表达。 第一个具体目标是识别和表征RARE 负责介导观察到的N-myc转录减少 在RA诱导的神经母细胞瘤细胞分化中。 这将是 通过使用瞬时转染的功能遗传分析完成 神经母细胞瘤细胞系的RA治疗前后。 此外，本发明还提供了一种方法， 使用凝胶阻滞和足迹法进行生化分析， 将这些序列的功能与特定的DNA相关联- 蛋白质结合 最后，将关键的核苷酸位置 RARE将通过观察特定突变对 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， RARE的研究将应用于细胞类型特异性沉默剂 来确定它的临界序列 调节蛋白结合 该序列将通过cDNA筛选来表征和克隆 用结合序列探针检测表达文库。 第三个具体目标是确定这些缺陷是否存在 调节区和/或DNA结合蛋白与 抗RA诱导分化，并与不利的 临床结果。 这将通过PCR扩增和测序进行检查 一组N-myc中的罕见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，以及在IV期N-myc 表达肿瘤（持续侵袭性，患者预后差）， 表达N-myc的IVS期肿瘤（最初转移，但经历了N-myc的转移） myc下调和分化为良性神经节细胞瘤），非 表达、分化的神经节细胞瘤和正常组织。 作为 沉默子结合蛋白产生的初始指示，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，以及在肿瘤和正常 组织. 这些知识应有助于患者的诊断， 分化剂在神经母细胞瘤治疗中的应用， 并提出了基于改变肿瘤细胞的新治疗方法， 通过调节致癌基因的表达来控制癌症的发生。