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.
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.
Kim
Eun J
EJ
Division of Life Sciences and Institute of Environmental and Life Sciences, Hallym University, Chunchon, Korea.
Schaffer
Beverly S
BS
Kang
Young-hee
YH
Macdonald
Richard G
RG
Park
Jung Hy
JH
D018938:Caco-2 Cells; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000473:pathology*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000037:antagonists & inhibitors* / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D018976:Insulin-Like Growth Factor Binding Protein 6; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II