ROLE OF METALLOTHIONEIN IN CANCER AND DRUG RESISTANCE

本研究将阐明一种重要的反式病毒的特征。 金属硫蛋白(MT)基因表达调控的激活因子 并确定该因子在细胞抵抗 镉毒性与肿瘤细胞获得性耐受性 顺铂等抗癌药物。我们的实验室已经描绘出一条26个BP 小鼠MT-I基因启动子区域的序列，命名为MRE-c‘， 这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 鉴定和提纯到接近均一的程度。代表这一点的cDNA克隆 蛋白质已经被分离出来。这项建议的四大具体目标 具体如下：(L)完成MBF2蛋白的鉴定 包括组织分布和任何翻译后修改 纯化的MBF-2蛋白，证实这些cDNA代表 获得全长MBF-2基因，并对其进行了全序列分析。 (2)调查那些自然表达的细胞和组织 基础水平越高的MT有更多的MBF-2，或者可能更活跃的MBF-2， 比其他人更好。这些实验将利用针对MBF-2的抗体和 报告基因构建比较MBF-2在顺铂敏感性中的作用 以及耐药细胞系。(3)调查MBF水平升高是否- 2的表达导致MT基因转录的基础水平升高 培养的细胞，如果是这样，是否增加了对顺铂和 镉被授予。MBF-2基因的表达将由一种 这些实验中无处不在的强大推动者。(4)使用转基因小鼠 直接检测MRE-c‘和MBF-2的重要性和功能。老鼠 过度表达这种基础转录因子最终可能证明 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可能受到 MT的表达水平。这项研究的长期目标是 描述MT表达所需的调节因子 基因，并探讨MT在镉诱导中的作用 毒性/肿瘤发生和细胞对金属的获得性抵抗力- 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.