Iron is an essential requirement for cellular proliferation. The metal gallium appears to inhibit cell growth by interfering with iron uptake and with iron-dependent steps in DNA synthesis. The long-term objectives of this proposal are: a) to study the interaction of gallium with iron metabolism and with the proteins of iron transport (transferrin and its receptor) and storage (ferritin); b) define factors involved in the uptake, intracellular localization and release of gallium from cells; c) isolate and characterize intracellular gallium-binding proteins; d) further study the mechanism of inhibition of DNA synthesis by gallium (which, in preliminary studies, appears to be due to its action on the iron-containing M2 subunit of ribonucleotide reductase). Although gallium is being used in clinical trials as an anti- neoplastic agent, little lead to a more judicious use of gallium in the treatment of specific malignancies, and may provide new information regarding iron requirements during cellular proliferation. Human leukemic K562 and HL60 cells adapted to long-term growth in serum-free media (with or without transferrin) will be used to study gallium uptake by transferrin-dependent versus transferrin- independent pathways. Studies will use gallium-67, dual-labeled 125I-transferrin-67Ga, and a fluorescent method for the detection of intracellular gallium. Agents which affect receptor internalization, receptosomal acidification, energy-dependent processes, and transferrin recycling will be used to define the role these steps play in gallium uptake. Cellular gallium-binding proteins will be isolated by gel filtration, ion-exchange chromatography and gel electrophoresis. Transferrin, transferrin receptor and ferritin will be immunoprecipitated by specific antibodies. Electron spin resonance spectroscopy and high performance liquid chromatography will be used to study the effect of gallium on ribonucleotide reductase (M2 tyrosyl radical) and nucleotide pools respectively; a specific assay will be used for DNA polymerase. Cell cycle analysis will be done by flow cytometry. Transferrin receptors shed from cells will be isolated by affinity chromatography, identified by gel electrophoresis, 125I-anti-receptor monoclonal antibody and autoradiography. 35S- methionine and 125I will be used to label and study the synthesis and release of the transferrin receptor from cells. It will be determined if the free transferrin receptor influences gallium cytotoxicity.
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