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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Hematology Subcommittee 2 (HEM)
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Medical College of Wisconsin
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Chitambar, C R; Wereley, J P (1995) Effect of hydroxyurea on cellular iron metabolism in human leukemic CCRF-CEM cells: changes in iron uptake and the regulation of transferrin receptor and ferritin gene expression following inhibition of DNA synthesis. Cancer Res 55:4361-6
Haq, R U; Wereley, J P; Chitambar, C R (1995) Induction of apoptosis by iron deprivation in human leukemic CCRF-CEM cells. Exp Hematol 23:428-32
Whelan, H T; Williams, M B; Bajic, D M et al. (1994) Gallium nitrate delays the progression of microscopic disease in a human medulloblastoma murine model. Pediatr Neurol 11:44-6
Whelan, H T; Williams, M B; Bajic, D M et al. (1994) Prevention of gallium toxicity by hyperhydration in treatment of medulloblastoma. Pediatr Neurol 10:217-20
Chitambar, C R; Wereley, J P; Riaz-ul-Haq (1994) Synergistic inhibition of T-lymphoblastic leukemic CCRF-CEM cell growth by gallium and recombinant human alpha-interferon through action on cellular iron uptake. Cancer Res 54:3224-8
Ul-Haq, R; Chitambar, C R (1993) Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells. Biochem J 294 ( Pt 3):873-7
Narasimhan, J; Antholine, W E; Chitambar, C R (1992) Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase. Biochem Pharmacol 44:2403-8
Chitambar, C R; Sax, D (1992) Regulatory effects of gallium on transferrin-independent iron uptake by human leukemic HL60 cells. Blood 80:505-11
Whelan, H T; Schmidt, M H; Anderson, G S et al. (1992) Antineoplastic effects of gallium nitrate on human medulloblastoma in vivo. Pediatr Neurol 8:323-7
Chitambar, C R; Narasimhan, J; Guy, J et al. (1991) Inhibition of ribonucleotide reductase by gallium in murine leukemic L1210 cells. Cancer Res 51:6199-201

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