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 proliferation. Because of the importance of IGF-binding proteins (IGFBPs) in the control of IGF activities that regulate cell proliferation in normal and malignant cell types, we undertook studies to characterize the IGFBPs produced by PC-3 prostate tumor cells in culture. We previously found, using an IGF-I affinity column for purification and a polyethylene 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 apparently not detected in the IGF-I-bound fraction with the PEG precipitation and Western ligand blot assays. In the current study, IGF-II affinity purification of IGFBPs produced by PC-3 cells, followed by C8 HPLC reverse-phase chromatography using a shallow acetonitrile gradient, 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 identical 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 125I-IGF-II/IGFBP-6 while it precipitated the complexes between 125I-IGF-II and other IGFBPs. Indeed, IGFBP-6 decreased the amount of 125I-IGF-II tracer in the PEG precipitate in a dose-dependent manner. Also, the binding of IGFBP-6 with 125I-IGF-II was poor in Western ligand blots compared with other IGFBPs. In studies on IGFBP-6 actions, IGFBP-6 completely inhibited IGF-II-induced [3H]thymidine incorporation in MC3T3-E1 mouse osteoblast cells while it had only minimal inhibitory effects on IGF-I-induced [3H]thymidine incorporation. This differential effect is associated with the fact that IGFBP-6 has greater affinity for IGF-II than IGF-I. The results of this study indicated that (1) Western ligand blotting is not sensitive 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 provide a useful model system for studying regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF actions.
Srinivasan
N
N
Department of Medicine, Loma Linda University, California 92357, USA.
Edwall
D
D
Linkhart
T A
TA
Baylink
D J
DJ
Mohan
S
S
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000302:isolation & purification; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D018974:Insulin-Like Growth Factor Binding Protein 4; D018976:Insulin-Like Growth Factor Binding Protein 6; D007335:Insulin-Like Growth Factor II