Gallium nitrate (NSC 15200) has significant clinical antineoplastic activity against lymphoma and bladder cancer, however, there is only a limited understanding of its mechanism of cytotoxicity and of tumor cell resistance to its growth-inhibitory effects. Our work has suggested that gallium's cytotoxicity is related to interference with cellular iron metabolism and ribonucleotide reductase (deoxyribonucleotide synthesis). This proposal has two Specific Aims: 1) To determine the mechanism of cytotoxicity of gallium and, 2) To investigate the basis of drug resistance to gallium.
The first aim will: a) determine the action of gallium on the transcription and translation of the transferrin receptor and ferritin genes (iron proteins) and, using an animal tumor model, test that hypothesis that the antitumor activity of gallium in vivo is due to perturbation of tumor iron metabolism; b) determine the mechanism by which gallium inhibits ribonucleotide reductase.
The second aim will: a) investigate the role of iron, transferrin, transferrin receptor, ferritin, iron-responsive element binding protein (lRE-BP, a transferrin receptor/ferritin mRNA-binding protein) and ribonucleotide reductase in the development of drug resistance to gallium and, b) examine if gallium- resistant cells are cross-resistant to other chemotherapeutic drugs and whether other drugs are synergistic with gallium. Human (HL60, CCRF-CEM, K562, others) and murine (L1210) cell lines will be used for in vitro experiments. mRNA levels, gene transcription and protein synthesis will be measured by Northern blotting, nuclear run-on assays and metabolic labeling of proteins. Protein-mRNA binding will be analyzed by band shift assay. Ribonucleotide reductase (its R1 and R2 subunits) will be studied by: enzyme assay, electron spin resonance (ESR) spectroscopy, and Western and Northern blotting. Deoxyribonucleotide pools will be measured by HPLC. Studies will use recombinant murine R2 protein and holoenzyme purified from cells by HPLC and ATP-agarose affinity chromatography. Human R1 and R2 will be studied using synthetic oligonucleotide probes and antibodies developed against human R1 and R2 peptides. Gallium resistance will be investigated using resistant cells developed by us. Transferrin receptors will be measured by ligand and antibody binding assays. 59Fe/ 67Ga -transferrin or -chelate complexes will be used to study iron/gallium uptake by cells. Immunoprecipitation, gel filtration, and gel electrophoresis will be used to identify gallium or iron associated with transferrin, transferrin receptor, ferritin, or ribonucleotide reductase. Transfected cells that overexpress H ferritin will be used to study the role of ferritin in gallium resistance. Cell proliferation and DNA synthesis assays will be used to evaluate synergy between gallium and other drugs. A tumor-bearing nude mouse model will be used for in vivo studies. Our studies will advance our understanding of gallium as a chemotherapeutic agent and will improve its use in the treatment of cancer.
