ROXY-16-ENE-23-YNE-26,27-HEXAFLUOROCHOLECALCIFEROL (RO24-5531) MODULATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 AND INDUCTION OF DIFFERENTIATION AND GROWTH ARREST IN A HUMAN OSTEOSARCOMA CELL-LINE

1 alpha,25-Dihydroxycholecalciferol [1,25-(OH)(2)D-3] is a potent diff erentiating agent in a variety of tumor cell lines. However, the induc tion of severe hypercalcemia has limited its clinical use. Several ana logs have been synthesized that retain the antiproliferative different iating effects of 1,25(OH)(2)D-3, but do not have the calcitropic effe ct of the parent compound. One such analog, 1 )(2)-16-ene-23-yne-26,27 -hexafluorocholecalciferol (Ro24-5531), can induce differentiation in HL-60 cells and does not induce hypercalcemia in animal models. We, th erefore, evaluated the effect of Ro24-5531 on a human osteosarcoma cel l Line, MG-63. Compared with 1,25-(OH)(2)D-3, the analog Ro24-5531 is 10-100 times more potent as an inhibitor of MG-63 cell proliferation, as determined by [H-3]thymidine incorporation and/or (4,5-dimethylthia zol-2-yl)-2,5-diphenyltetrazolium bromide assay. The inhibition in cel l growth is accompanied by a decrease in the expression of p34cdc2 (>4 -fold), a protein critically involved in cell cycle regulation. Ro24-5 531 treatment of MG-63, at a concentration of 10(-8) mol/L, induced ex pression of the bone differentiation markers biglycan and osteocalcin, as determined by Northern analysis. These data suggest that Ro24-3531 treatment induces growth arrest coupled with differentiation. To begi n to evaluate the mechanisms by which Ro24-5531 may exert an effect, w e evaluated the effect of Ro24-5531 on components of the insulin-like growth factor I (IGF-II signaling pathway, an important regulator of n ormal bone growth and differentiation. The expression of IGF-binding p rotein (IGFBP), IGFBP-3 messenger ribonucleic acid, and protein levels are increased 20-fold after 72 h of treatment with Ro24-5531 and are associated with a marked increase in detectable binding of ligand to b inding protein, as measured by RRA. These data suggest an association between Ro24-5531-induced growth arrest and increased expression of IG FBP-3.