ROLE OF METALLOTHIONEIN IN CANCER AND DRUG RESISTANCE

本研究将阐明一个重要的跨- 激活因子控制金属硫蛋白（MT）基因表达， 并确定该因子在细胞抵抗 镉毒性和肿瘤细胞对强毒性镉的获得性抗性 抗癌药物如顺铂。我们的实验室已经描绘出了一个26 bp的 小鼠MT-1基因启动子区的序列，命名为MRE-c ′， 是基础转录所必需的。一种新的蛋白质因子MBF-2， 与该序列相互作用并控制MT基因表达的基因， 鉴定并纯化至接近同质。代表这一点的cDNA克隆 蛋白质已被分离。该提案的四大具体目标 完成MBF 2蛋白的鉴定 包括组织分布和任何翻译后修饰 纯化的MBF-2蛋白，证实cDNA代表了 全长MBF-2 mRNA，cDNA全序列分析。 (2)研究那些自然表达的细胞和组织 MT的基础水平越高，MBF-2越多，或者可能更活跃， than others.这些实验将利用针对MBF-2的抗体和抗MBF-2抗体。 用于比较MBF-2在顺铂敏感性中功能的报告基因构建体 和抗性细胞系。(3)调查是否增加了MBF水平- 2的表达导致MT基因转录的基础水平更高， 培养的细胞，如果是这样，是否增加了对顺铂的耐药性， 镉被赋予。MBF-2 cDNA的表达将由一个启动子驱动。 强的普遍存在的启动子。(4)使用转基因小鼠 直接测试MRE-c ′和MBF-2的重要性和功能。小鼠 过度表达这种基础转录因子最终可以证明 可用作测试抗性生理方面的模型系统 其他抗癌药物和有毒化合物， MT表达水平。这项研究的长期目标是 表征MT表达所需的调节因子 基因，并探讨MT在镉诱导的 毒性/肿瘤发生和获得性细胞对金属的抗性， 含有抗癌药物。

This study will elucidate the characteristics of an important trans- activating factor in the control of metallothionein (MT) gene expression, and determine the potential role of this factor in cellular resistance to cadmium toxicity and the acquired resistance of tumor cells to potent anticancer drugs such as cisplatin. Our laboratory has delineated a 26 bp sequence in the promoter region of the mouse MT-I gene, designated MRE-c', that is required for basal transcription. A novel protein factor, MBF-2, that interacts with this sequence and controls MT gene expression has been identified and purified to near-homogeneity. cDNA clones representing this protein have been isolated. The four major specific aims of this proposal are as follows: (l) Complete the characterization of the MBF2 protein including tissue distribution and any post-translational modifications of the purified MBF-2 protein, confirmation that the cDNAs represent the full-length MBF-2 mRNA, and the complete sequence analysis of the cDNA. (2) Investigate whether those cells and tissues that naturally express higher basal levels of MT have more MBF-2, or perhaps more active MBF-2, than others. These experiments will utilize antibodies to MBF-2 and a reporter gene construct to compare MBF-2 function in cisplatin-sensitive and resistant cell lines. (3) Investigate whether increased levels of MBF- 2 expression lead to higher basal levels of MT gene transcription in cultured cells and if so, whether an increased resistance to cisplatin and cadmium is conferred. Expression of the MBF-2 cDNA will be driven from a strong ubiquitous promoter in these experiments. (4) Use transgenic mice to test directly the importance and function of MRE-c' and MBF-2. Mice which overexpress this basal transcription factor could ultimately prove useful as a model system for testing physiological aspects of resistance to other anti-cancer drugs and toxic compounds that might be influenced by MT expression levels. The long-term objectives of this research are to characterize the regulatory factors required for expression of the MT genes, and explore the role of MT in cadmium-induced toxicity/tumorigenesis and acquired cellular resistance to metal- containing anticancer drugs.