REGULATION OF THE N MYC ONCOGENE IN NEUROBLASTOMA

N MYC 癌基因在神经母细胞瘤中的调控

• 批准号: 2735674
• 负责人: RANDAL K WADA
• 金额: $ 20.1万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2735674
• 关键词: DNA footprinting; DNA methylation; HeLa cells; affinity chromatography; chimeric proteins; complementary DNA; differentiation antigens; gel mobility shift assay; gene induction /repression; gene mutation; genetic promoter element; molecular cloning; neoplasm /cancer genetics; neuroblastoma; nucleic acid sequence; oligonucleotides; oncogenes; polymerase chain reaction; retinoate; site directed mutagenesis; thymidine kinase; transcription factor

The broad objectives of this proposal are to identity 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 clement, 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 differences from the silencer binding regulatory proteins 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 disease in N-myc transcription observed upon RA induced differentiation of neuroblastoma cells. This region has been mapped by functional genetic analysis using transient transfections of neuroblastoma cell lines before and alter RA treatment. Biochemical analyses using gel retardation and footprinting will be performed to correlate function of these sequences with specific DNA-protein binding. The critical nucleotide positions within the RARE will then be determined by observing the effect of specific mutations on RARE function and protein binding. The second specific aim is to identity the DNA sequence and protein(s) mediating the silencing of the N-myc promoter in non-neuroblastoma cells. Using N-myc expressing neuroblastoma, and non-expressing, nonneuroblastoma 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 by weaning of a cDNA expression library with the binding sequence probe. The third specific aim is to determine whether defects 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 (persistently aggressive with poor patient outcome), N- myc expressing stage IVS tumors (initially metastatic, but undergo N-myc down regulation and differentiation to benign glioneuroma), 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 oncogene expression.

这项提案的广泛目标是确定DNA控制 调控N-myc启动子活性的元件 人类神经母细胞瘤中的癌基因，并确定错配是否在 这一规律具有临床意义。初步研究表明 确定N-myc启动子中介导其下调的区域 维甲酸(RA反应温和，或罕见)，以及其在非 表达的非神经母细胞瘤细胞。此外，这些结果还包括 表明在某些情况下，罕见的突变可能是 对RA的抗性诱导分化，而不同于 非表达细胞中存在的沉默分子结合调节蛋白可能 导致神经母细胞瘤中N-myc的结构性基础表达。 第一个具体目标是识别和描述稀有的 负责介导N-myc转录中观察到的疾病 RA诱导神经母细胞瘤细胞分化。这一地区一直是 利用瞬时转染法进行功能遗传分析 RA治疗前后神经母细胞瘤细胞株的建立。生化 使用凝胶延迟和足迹分析将执行 这些序列的功能与特定的DNA-蛋白质结合有关。 然后将确定RARE中的关键核苷酸位置 通过观察特定突变对稀有功能和蛋白质的影响 有约束力的。 第二个特定目的是鉴定DNA序列和蛋白质(S) 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的神经母细胞瘤和非表达的非神经母细胞瘤 细胞系，遗传和生化分析在研究中的应用 Rare将应用于细胞类型特定的消音器以确定其 关键顺序。调节蛋白(S)将与此序列结合 特征，并通过断奶表达文库克隆 结合序列探针。第三个具体目标是确定是否 这些调节区和/或dna结合蛋白的缺陷(S)是 与对RA诱导分化的抗性有关，并与 有不良的临床结果。这将通过聚合酶链式反应扩增和 对一组N-的稀有和细胞类型特定的消音器元件进行测序 MYC表达，RA敏感和耐药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(持续侵袭性，患者预后差)，N- MYC表达的IVS期肿瘤(最初转移，但经历N-myc 下调和分化为良性神经胶质瘤)，非 表达、分化的神经节细胞瘤和正常组织。作为一种 消音器结合蛋白产生的初步迹象，反向 转录聚合酶链式反应将被用来定量其在N-myc中的mRNA表达 表达和不表达的细胞系，以及在肿瘤和正常 组织。 这样的知识应该有助于患者的预测，提供对 分化药物在神经母细胞瘤治疗中的应用，以及 基于改变肿瘤细胞行为提出新的治疗方法 通过调节癌基因的表达。