CLONING OF THE FUNCTIONAL PROMOTER FOR HUMAN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-4 GENE - ENDOGENEOUS REGULATION

The majority of the colon cancers analyzed to-date express insulin-lik e growth factor binding protein (IGFBP)-4, and antisense inhibition of IGFBP-4 messenger RNA (mRNA) confers a growth advantage to the cells in response to endogenous and exogenous IGFs. We recently reported a s ignificant up-regulation of IGFBP-4 expression in a human colon cancer cell line (CaCo2) on spontaneous differentiation of the cells in cult ure. This suggests that the expression of IGFBP-4 may be located to gr owth and differentiation of colon cancer cells. To study the endogenou s factors involved in the transcriptional regulation of IGFBP-4, we ha ve isolated and sequenced the human (h) IGFBP-4 promoter. The approxim ately 1.3 kilobase pair (kb) 5' flanking region of the ICFBP-4 gene is GC rich and possesses several potential regulatory element. These ele ments include a typical TATA box with sequence TATAA, located -299 nt from the initiation ATG codon. The cap site is located 14 nt downstrea m of the TATA box as determined by primer extension analysis. A 1.4-kb DNA fragment including the 1.264 kb 5' flanking region of the hIGFBP- 4 gene was subcloned into a luciferase reporter vector (pGL-2 basic) e ither in the sense (BP-4-S-pGL) (S) or antisense (BP-4-AS-pGL) (AS) (n egative control) orientation, relative to the luciferase coding sequen ce in the vector. CaCo2 cells were transfected with either the S or th e AS vectors on days 2-10 of culture; cotransfection with the SV40-bet a-Galactidose (Gal) vector was used to correct for transfection effici ency. The ratio of luciferase/beta-Gal expression by CaCo2 cells trans fected with the S vectors increased significantly from days 3 and 4 to days 5 and 6 of culture, followed by a sharp decline on days 7-9, res embling the pattern of endogenous expression of IGFBP-4 by the cells; the expression of luciferase by the AS vectors remained low and insign ificant. These results thus suggest that the approximately 1.4 kb 5' f lanking region of the IGFBP-4 gene contains the cia elements required for regulation of the IGFBP-4 gene. Cloning and sequencing of the func tional hIGFBP-4 promoter will enable us, for the first time, to study the endogenous factors/mechanisms responsible for the growth/different iation (cell density) associated regulation of lGFBP-4 expression in c olonic epithelial cells.