This research project involves the generation of mammalian cells which are defective in the down-regulation of the transferrin receptor (tfr) in response to iron. Iron, which normally functions as an enzyme cofactor is a critical nutrient, but one which can be extremely toxic if overabundant. Iron poisoning is the leading cause of death in young children in the United States. In the industrialized countries, overabundance of dietary iron has been implicated in cancer and heart disease. Iron is believed to cause disease via the generation of highly reactive free radical oxygen species which, even in small quantities can destroy the critical cellular activities of proteins, enzymes and lipids. In the developing world where iron deficiency anemia is a major health problem, dietary iron is insufficient to meet critical cellular requirements for enzymes such as ribonucleotide reductase, which is required for the synthesis of DNA for cell division. The tfr is one of the two major cellular proteins responsible for maintaining the narrow limits of iron homeostasis required for life. My research plan, the generation and characterization of mammalian cells mutant in the ability to regulate tfr levels, will allow us to understand how mammalian cells regulate intracellular iron levels. During FY94, we have been able to develop a method for separating cells cultured in iron-rich vs. iron- depleted environments. We have successfully used fluorescence-activated cell sorting (FACS) to prepare tfr-rich or tfr-poor cell populations. In this method, cells are labeled with fluorescent transferrin, which labels both the membrane bound and intracellular cycling pool of tfr~s. This allows us to re-culture these cells after FACS, so that they may be subjected to additional rounds of FACS in order to homogenize the population and study them. We have successfully generated an initial population of cultured cells which are enriched in clones unable to down- regulate the tfr in response to environmental iron via mutagenesis with methane sulfonic acid ethyl ester. We plan to use FACS to obtain a homogeneous population of mutants of this class, generate clonal populations and analyze the phenotype of cells unable to prevent iron uptake via the tfr. Since tfr mediated iron uptake is the major pathway for iron entry into higher eukaryotic cells, we expect these mutants to be profoundly affected at the levels of cell division, membrane integrity, electron transport and to have other as yet uncharacterized defects. Ultimately, the rescue of such mutants and cloning of genes responsible for the rescue of these mutant phenotypes should allow us to identify the polypeptide and possibly nucleic participants in the down- regulation of the tfr in response to iron. This down-regulation is accomplished by increased degradation of the tfr mRNA. Consequently, RNA half-life must be quantified in order to characterize mutant cells. To this end, we have successfully cloned the appropriate cDNA and developed a sensitive RNase Protection Assay for tfr mRNA.
