INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-6 PRODUCED BY HUMAN PC-3 PROSTATE-CANCER CELLS - ISOLATION, CHARACTERIZATION AND ITS BIOLOGICAL ACTION

The PC-3 human prostatic carcinoma cell line has been extensively used as a model for studies on the regulation of prostate tumor cell proli feration. Because of the importance of IGF-binding proteins (IGFBPs) i n the control of IGF activities that regulate cell proliferation in no rmal and malignant cell types, we undertook studies to characterize th e IGFBPs produced by PC-3 prostate tumor cells in culture. We previous ly found, using an IGF-I affinity column for purification and a polyet hylene glycol (PEG) precipitation assay for IGFBP detection, that PC-3 cells in culture produced a single predominant IGFBP, IGFBP-4, which inhibits IGF activities. We now present evidence that PC-3 cells also produce IGFBP-6 in abundant quantity; in the previous study this was a pparently not detected in the IGF-I-bound fraction with the PEG precip itation and Western ligand blot assays. In the current study, IGF-II a ffinity purification of IGFBPs produced by PC-3 cells, followed by C8 HPLC reverse-phase chromatography using a shallow acetonitrile gradien t, produced two major protein peaks. N-terminal amino acid sequence of peak 1 was identical to that of IGFBP-6 while that of peak 2 was iden tical to that of IGFBP-4. Characterization of purified IGFBP-6 from PC -3 cells revealed properties which are distinct from other IGFBPs. PEG did not precipitate the complex of I-125-IGF-II/IGFBP-6 while it prec ipitated the complexes between I-125-IGF-II and other IGFBPs. Indeed, IGFBP-6 decreased the amount of I-125-IGF-II tracer in the PEG precipi tate in a dose-dependent manner. Also, the binding of IGFBP-6 with I-1 25-IGF-II was poor in Western Ligand blots compared with other IGFBPs. In studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II-in duced [H-3]thymidine incorporation in MC3T3-E1 mouse osteoblast cells while it had only minimal inhibitory effects on IGF-I-induced [3H]thym idine incorporation. This differential effect is associated with the f act that IGFBP-6 has greater affinity for IGF-II than IGF-I. The resul ts of this study indicated that (1) Western ligand blotting is not sen sitive for identification of IGFBP-6, (2) the unique behavior of IGFBP -6 in the PEG assay system necessitates the use of charcoal adsorption procedure for IGFBP-6 activity detection and (3) PC-3 cells should pr ovide a useful model system for studying regulation of IGFBP-6 express ion and the role of IGFBP-6 in modulating IGF actions.