The long-term objectives and specific aims of the projects in this research proposal are 1) to obtain a better understanding of cellular iron requirements during cellular proliferation and to define the interaction of gallium with cellular iron metabolism since gallium appears to inhibit cell growth by interfering with cellular iron utilization in an unknown manner and has been used in clinical cancer trials as a cytotoxic agent. An understanding of this interaction may provide new information regarding cellular requirements for iron during proliferation and information as to how these requirements may be exploited to control tumor cell growth. 2) To establish fibroblast cultures from patients with Hemochromatosis (a disease characterized by iron-overload resulting in multi-organ damage) and study transferrin receptor regulation and factors influencing iron uptake in these cells. The studies may elucidate new information regarding this disease. Studies of the effect of gallium on intracellular distribution of 59Fe will include gel filtration of solubilized cytoplasmic contents and immunoprecipitation of the peak radioactive fractions for transferrin, transferrin receptor and ferritin using specific antisera. Subcellular fractionation will also be performed using sucrose gradient centrifugation. Cellular isoferritins will be studied during gallium-controlled cell proliferation using isoelectric focusing. Studies of 67gallium uptake and intracellular distribution will be performed using similar methodology. Gallium-binding proteins will be identified by gel electrophoresis. Studies of cellular receptosome unloading of iron in gallium treated cells will employ studies of receptosomal pH changes using FITC-labeled transferrin-metal probes and analysis in a spectrofluorometer. Studies of effects of different transferrin-metals on receptor cycling/recycling will include 59Fe/125I-transferrin in pulse-chase experiments. Studies of transferrin receptor synthesis and phosphorylation will employ 35S-methionine and 32P labeling of the receptor with isolation by affinity chromatography. 32P labeled phosphorylated transferrin receptor will be identified on gels using autoradiography. The different transferrin-metals, iron chelators and antitransferrin receptor antibodies will be used to study transferrin receptor regulation and iron uptake in fibroblasts from patients with Hemochromatosis. Transferrin receptors will be assayed using 125I-transferrin binding, radioimmunoassay, and measurement of immunofluorescent cell surface transferrin receptor with a fluorocytometer.
| Chitambar, C R; Zivkovic, Z (1987) Inhibition of hemoglobin production by transferrin-gallium. Blood 69:144-9 |
