IRON REGULATION OF HUMAN TRANSFERRIN SYNTHESIS

描述：待检验的假设是，一个假定的铁反应性 通过与其他三种mRNA中的序列同源性鉴定的元件 （铁蛋白、转铁蛋白受体和红系氨基乙酰丙酸合酶） 在抑制转铁蛋白mRNA的表达中起作用， 对铁水平升高的反应。 具体目标首先是建立 铁的调节作用在转基因小鼠中丧失， 嵌合人转铁蛋白氯霉素乙酰转移酶转基因 其中IRE的突变导致蛋白结合的丧失， 体外;第二，在培养的肝癌细胞（HepG 2）中测定 转铁蛋白的时程和铁的调节;第三， 适当突变的转铁蛋白-CAT构建体的瞬时转染， 在人肝细胞的模型系统中建立响应;四， 使用电泳技术表征体外蛋白质-RNA相互作用 纯化的IRP（已经证明）或细胞质的迁移率变化测定 Hep G2细胞提取物，第五，在转基因小鼠中扩展研究 其表达完整的野生型人转铁蛋白或产生人转铁蛋白 mRNA与突变的IRP和不再是铁响应性的，并决定 铁处理对人转铁蛋白组织和血清水平的影响。 研究设计是适当的，并使用人类和小鼠实验 系统，包括需要突变的复杂技术， 转基因CAT构建体，瞬时转染，现有菌株， 转基因小鼠具有与无转铁蛋白血症小鼠杂交的潜力。 的 这项研究的意义在于，它将最终建立一个 铁下调转铁蛋白表达的机制， 提供了转铁蛋白合成机制的新信息， 可以用来设计更有效的癌症治疗方法， 转铁蛋白被用作毒性剂如镓或 共价连接的配体如多柔比星，以将这些试剂靶向 表达大量表面转铁蛋白的快速生长的肿瘤细胞 受体。 尽管PI在申请中实际上没有强调， 对于未来的研究，事实是该元素位于5'区域， 转铁蛋白mRNA和铁蛋白，但有相反的影响，调节 对铁的反应。

DESCRIPTION: The hypothesis to be tested is that a putative iron-responsive element identified by sequence homology with those in three other mRNAs (ferritin, transferrin receptor and erythroid amino-levulinate synthase) functions in the suppression of expression of the transferrin mRNA in response to raised iron levels. The specific aims are first to establish that regulation by iron is lost in transgenic mice expressing a mutated chimeric human transferrin chloramphenicol acetyltransferase (CAT) transgene in which the mutation of the IRE results in loss of protein binding in vitro; second, to determine in cultured hepatoma (HepG2) cells determine time course and regulation by iron of transferrin; and third, using transient transfection of appropriately mutated transferrin-CAT constructs, to establish the response in a model system for human liver cells; four, to characterize protein-RNA interactions in vitro using electrophoretic mobility shift assays of purified IRP (already demonstrated) or cytoplasmic extracts of Hep G2 cells, and fifth, to extend studies in transgenic mice which express intact wildtype human transferrin or produce human transferrin mRNA with mutated IRP and are no longer iron-responsive and determine the effect of iron treatment on tissue and serum levels of human transferrin. The research design is appropriate and uses human and murine experimental systems including sophisticated technologies requiring mutation of transgenic CAT constructs, transient transfection, existing strains of transgenic mice with the potential to cross with atransferrinemic mice. The significance of this research is that it will definitively establish a mechanism whereby transferrin expression is down-regulated by iron and provides new information on the mechanisms of transferrin synthesis which can then be applied to design more effective therapies for cancer, in which transferrin is used as a carrier of toxic agents such as gallium or covalently linked ligand such as doxorubicin to target these agents to rapidly growing tumor cells which express abundant surface transferrin receptors. Although not actually stressed by the PI in the application or for future studies, the fact is that the element resides in the 5' region of the transferrin mRNA and of ferritin but has opposite effects on regulation in response to iron.