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 an d only one of the six IGF-binding proteins, IGFBP-4, In the present st udy, the physiological role of endogenous IGFBP-4 in regulating the gr owth response of HT-29 cells to exogenous and endogenous IGFs was exam ined, 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 f urther confirm the inhibitory role of endogenous IGFBP-4, sense and an tisense complementary DNA fragments of human IGFBP-4 mere 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 di rections in the pCEP4 vectors, HT-29 cells were transfected with eithe r the control (no insert, C-P), sense (S-P), or antisense (AS-P) vecto rs and subjected to hygromycin selection, The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in th e conditioned media (CM) of 10(7) cells by ligand and immunoblot analy sis, 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 conce ntrations 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-stimulate d growth of the AS-P cells was significantly higher than that of the C -P and S-P cells, The basal (nonstimulated) and the IGF-I-stimulated g rowth of the S-P cells was not significantly different from that of th e C-P cells, suggesting that overexpression of IGFBP-4 was not inhibit ory 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 anti body, once again suggesting that endogenous IGFBP-4 was a potent inhib itor 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 differ ence in the endogenous secretion of IGFBP-4 by the cells, In the prese nt study, we have, for the first time, demonstrated a potent inhibitor y role of endogenous IGFBP-4 for regulating the mitogenic response of the cells to both endogenous and exogenous IGFs. An important observat ion 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 ad dition of IGFBP-4 antibody, on the other hand, could effectively remov e this block, The latter findings suggest that IGFBP-4 may be inhibiti ng the mitogenic effects of IGF-I by other possible IGF-independent me chanisms.