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Characterization of the inhibition of DNA synthesis in proliferating mink lung epithelial cells by insulin‐like growth factor binding protein‐3
Author(s) -
Wu HaiBin,
Kumar Amit,
Tsai WenChi,
Mascarenhas Desmond,
Healey Judy,
Rechler Matthew M.
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(20000501)77:2<288::aid-jcb11>3.0.co;2-j
Subject(s) - incubation , dna synthesis , growth factor , mink , endogeny , biology , insulin like growth factor binding protein , insulin like growth factor , microbiology and biotechnology , receptor , cell culture , cell growth , dna , biochemistry , ecology , genetics
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 ∼400‐kDa complex. This complex co‐migrated with a complex formed after incubation with 125 I‐labeled transforming growth factor‐β (TGF‐β) that has been designated the type V TGF‐β receptor. 125 I‐IGFBP‐3 binding to the ∼400‐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 ∼400‐kDa receptor is consistent with the hypothesis that the ∼400‐kDa IGFBP‐3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP‐3. J. Cell. Biochem. 77:288–297, 2000. © 2000 Wiley‐Liss, Inc.