title: Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I.
abstract: HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 10(7) cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 +/- 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555.
authors.forename: P
authors.initials: P
authors.lastname: Singh
authors.forename: B
authors.initials: B
authors.lastname: Dai
authors.forename: B
authors.initials: B
authors.lastname: Dhruva
authors.forename: S G
authors.initials: SG
authors.lastname: Widen
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D003110:Colonic Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D016373:DNA, Antisense / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D016373:DNA, Antisense; D018076:DNA, Complementary; D007328:Insulin; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I