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: 2273665
• 负责人: RANDAL K WADA
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2273665
• 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.

该提案的主要目标是识别 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，并确定是否存在紊乱 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），及其在非 表达的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 诱导分化的抗性，以及与 非表达细胞中存在的沉默子结合调节蛋白可能 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 负责介导在 N-myc 转录中观察到的疾病 RA诱导神经母细胞瘤细胞分化。该地区已 使用瞬时转染的功能遗传分析绘制图谱 RA 治疗前和治疗后的神经母细胞瘤细胞系。生化 将使用凝胶延迟和足迹进行分析 将这些序列的功能与特定的 DNA-蛋白质结合联系起来。 然后将确定 RARE 中的关键核苷酸位置 通过观察特定突变对 RARE 功能和蛋白质的影响 绑定。 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 的非神经母细胞瘤 研究中使用的细胞系、遗传和生化分析 RARE 将应用于细胞类型特定的消音器，以确定其 关键序列。与该序列结合的调节蛋白将是 通过断奶 cDNA 表达文库进行表征和克隆 结合序列探针。第三个具体目标是确定是否 这些调节区域和/或 DNA 结合蛋白的缺陷是 与对 RA 诱导分化的抗性相关，并相关 具有不良的临床结果。这将通过 PCR 扩增进行检查 对一组 N- 中的 RARE 和细胞类型特异性沉默元件进行测序 表达 myc、RA 敏感和耐药的神经母细胞瘤细胞系 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），N- myc 表达 IVS 期肿瘤（最初是转移性的，但经历 N-myc 下调并分化为良性胶质神经瘤），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预测，提供洞察力 分化剂在神经母细胞瘤治疗中的应用，以及 提出基于改变肿瘤细胞行为的新治疗方法 通过调节癌基因的表达。