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

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 107 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-H). 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. In the present study, we have, for the first time, demonstrated a potent inhibitory role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observation was that the inhibitory effects of endogenous IGFBP-4 could not be titrated out by the addition of an excess of IGF-I or insulin; the addition of IGFBP-4 antibody, on the other hand, could effectively remove this block. The latter findings suggest that IGFBP-4 may be inhibiting the mitogenic effects of IGF-I by other possible IGF-independent mechanisms.