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Decreased production of insulin‐like growth factor‐binding protein (IGFBP)‐6 by transfection of colon cancer cells with an antisense IGFBP‐6 cDNA construct leads to stimulation of cell proliferation
Author(s) -
KIM EUN J,
SCHAFFER BEVERLY S,
KANG YOUNGHEE,
MACDONALD RICHARD G,
PARK JUNG HY
Publication year - 2002
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1046/j.1440-1746.2002.02703.x
Subject(s) - transfection , clone (java method) , microbiology and biotechnology , cell growth , growth factor , biology , cell culture , insulin like growth factor , sense (electronics) , western blot , insulin like growth factor binding protein , northern blot , complementary dna , receptor , gene , chemistry , biochemistry , genetics
Background: Previously, we have observed that highly unsaturated dietary (n‐3) fatty acids inhibit cell proliferation in conjunction with stimulation of insulin‐like growth factor‐binding protein (IGFBP)‐6 secretion in Caco‐2 cells, a human colon carcinoma cell line.Methods: To test the converse hypothesis that inhibition of endogenous IGFBP‐6 secretion stimulates Caco‐2 cell proliferation, cells were transfected with the antisense IGFBP‐6 expression construct or pcDNA3 vector only, and single colonies resistant to G418 sulfate were isolated.Results: Our initial studies indicated that three antisense clones grew faster and produced less IGFBP‐6 than two pcDNA3 clones, so antisense IGFBP‐6 #5 and pcDNA3 #8 were selected for further detailed analysis. Both the control and antisense clones grew in serum‐free medium reaching a plateau density at day eight. However, the antisense clone grew at a rate faster than that of the control and reached a final density that was 31 ± 3% higher than the control. Northern blot, ligand blot and immunoblot analyses revealed that accumulation of IGFBP‐6 mRNA and concentrations of IGFBP‐6 peptide produced by the antisense clone were decreased by 80–90% compared to the control. The doubling times of the antisense and control clones were 21.9 ± 0.4 and 24.8 ± 0.3 h ( P < 0.05), respectively. Exogenous IGF‐I and IGF‐II (0.2–200 nmol/L) stimulated proliferation of both the control and antisense clones in a dose‐dependent manner, but the relative potency and efficacy of IGF‐II was higher in the antisense clone compared to the control. These results indicate that suppression of IGFBP‐6 secretion correlates with an increase in the basal rate of Caco‐2 cell growth.Conclusions: Our findings are consistent with the hypothesis that IGFBP‐6 inhibits cell growth by binding to endogenously produced IGF‐II, thereby preventing IGF‐II from interacting with the IGF‐I receptor to stimulate cellular proliferation by an autocrine mechanism. © 2002 Blackwell Publishing Asia Pty Ltd