title: Characterization of the inhibition of DNA synthesis in proliferating mink lung epithelial cells by insulin-like growth factor binding protein-3.
abstract: Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.
authors.affiliation: Growth and Development Section, Clinical Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1758, USA.
authors.forename: H B
authors.initials: HB
authors.lastname: Wu
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: W C
authors.initials: WC
authors.lastname: Tsai
authors.forename: D
authors.initials: D
authors.lastname: Mascarenhas
authors.forename: J
authors.initials: J
authors.lastname: Healey
authors.forename: M M
authors.initials: MM
authors.lastname: Rechler
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D007334:Insulin-Like Growth Factor I; D004247:DNA