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Induction of Apoptosis in Human Prostate Cancer Cells by Insulin-Like Growth Factor Binding Protein-3 Does Not Require Binding to Retinoid X Receptor-

Diabetes Branch (G.Z., J.E., M.M.R., N.B.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and Department of Cancer Pathways (C.E., J.G.), Merck Research Laboratories, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Matt Rechler, National Institutes of Health, Building 10, Room 8D12, 9000 Rockville Pike, MSC 1758, Bethesda, Maryland 20892. E-mail: mrechler{at}helix.nih.gov.

IGF binding protein (IGFBP)-3 can induce apoptosis in human prostate cancer cells directly without sequestering IGF-I and -II. The molecular mechanisms responsible for the IGF-independent actions of IGFBP-3 remain unclear. IGFBP-3, a secreted protein, can be internalized and translocate to the nucleus. It binds to the nuclear retinoid X receptor (RXR)-. Binding to RXR- has been proposed to be required for IGFBP-3 to induce apoptosis. The present study tests this hypothesis in the PC-3 human prostate cancer cell line. PC-3 cells express RXR-, and apoptosis is induced by incubation with RXR-specific ligand. A COOH-terminal region in IGFBP-3 (residues 215–232) contains a nuclear localization signal, and binding domains for RXR- and heparin (HBD). Different combinations of the 11 amino acids in this region that differ from IGFBP-1, a related IGFBP, which does not localize to the nucleus or bind RXR-, were mutated to the IGFBP-1 sequence. By confocal imaging, mutation of residues 228-KGRKR-232 in nonsecreted IGFBP-3 diminished its nuclear localization. IGFBP-3 binding to glutathione S-transferase-RXR- only was lost when all 11 sites were mutated (HBD-11m-IGFBP-3). Expressed nuclear RXR- did not transport cytoplasmic IGFBP-3 nuclear localization signal mutants that can bind RXR- to the nucleus even after treatment with RXR ligand. Expressed HBD-11m-IGFBP-3 still induced apoptosis in PC-3 cells in an IGF-independent manner as determined by flow cytometric analysis of Annexin V staining. We conclude that in PC-3 cells, RXR- is not required for the nuclear translocation of IGFBP-3 and that IGFBP-3 can induce apoptosis in human prostate cancer cells without binding RXR-.