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.
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