Regulation of the N-myc oncogene in neuroblastoma

描述：（由申请人提供）本提案的目标是确定 神经母细胞瘤细胞N-myc基因转录下调机制的研究 维甲酸（RA）。RA通过转录途径对N-myc发挥转录作用 启动子的大致限定区域（RA反应区，或RARR）。这些 研究将检验N-myc表达细胞中RARR突变的假设， 导致转录因子的互补或构型的改变 与启动子结合，导致不能下调N-myc RA处理后，细胞对RA诱导的表型抗性降低， 分化，以及缺乏对RA的临床反应。在第一个具体的 目的，瞬时转染和DNA凝胶转移，交联和足迹 分析将用于鉴定和表征启动子序列， 基础N-myc转录所必需的DNA结合蛋白， 通过RA下调，以及其在RA抗性细胞中的组成型表达。在 第二个具体目标，阻断RA的突变的生物学效应， 启动子的诱导下调将使用稳定的 含有由野生型或突变启动子驱动的N-myc的转染子。 第三个具体目标将决定RARR的临床意义 通过比较它们在RA敏感和耐药细胞中的发生率， 在诊断时和RA治疗后复发后获得的肿瘤对中， 以及高风险和低风险肿瘤。诊断时存在RARR突变 也将与高风险人群的生存率降低相关。 接受RA治疗的神经母细胞瘤患者。RA是目前唯一有效的 用于治疗神经母细胞瘤患者的生物改性剂 残留病从这些调查中获得的结果将提供 基于分子的机械基础， 关于RA治疗的患者说明。这些数据将 帮助确定RA耐药患者候选人，用于其他试验 生物活性剂。最终，对癌基因调控的理解 有利于未来合理发展组合分化 神经母细胞瘤的治疗，这可能是有效的，即使在诱导 防止大体积疾病。

DESCRIPTION: (provided by applicant) The goal of this proposal is to identify the mechanism of N-myc transcriptional downregulation in neuroblastoma cells by retinoic acid (RA). RA exerts its transcriptional effects on N-myc through a roughly defined region of the promoter (RA response region, or RARR). These studies will test the hypothesis that RARR mutations in N-myc expressing cells leads to alteration of the complement or configuration of transcription factors that bind to the promoter, resulting in the inability to downregulate N-myc after RA treatment, the phenotypic resistance of cells to RA-induced differentiation, and the lack of clinical response to RA. In the first specific aim, transient transfections and DNA gel shift, cross linking, and footprinting analyses will be used to identify and characterize the promoter sequences and DNA binding proteins necessary for basal N-myc transcription, its downregulation by RA, and its constitutive expression in RA resistant cells. In the second specific aim, the biological effects of mutations that block the RA induced downregulation of the promoter will be tested using stable transfectants containing N-myc driven by either wild type or mutated promoters. The third specific aim will determine the clinical significance of RARR mutations by comparing their incidence in RA sensitive and resistant cell lines, in tumor pairs obtained at diagnosis and after relapse post-RA therapy, and in high and low risk tumors. The presence of RARR mutations at diagnosis will also be correlated with decreased survival in a population of high-risk neuroblastoma patients treated with RA. RA is currently the only effective biologic modifier for the therapy of neuroblastoma patients with minimal residual disease. Results obtained from these investigations will provide a molecular-based, mechanistic foundation that will allow eventual prognostication of patients with respect to treatment with RA. Such data will aid in identifying RA-resistant patient candidates for trials of other biologically active agents. Ultimately, an understanding of oncogene regulation will facilitate the future rational development of combination differentiation therapy of neuroblastoma, which might be effective even in the induction setting against bulky disease.