PROLIFERATION AND DIFFERENTIATION OF A HUMAN COLON-CANCER CELL-LINE (CACO2) IS ASSOCIATED WITH SIGNIFICANT CHANGES IN THE EXPRESSION AND SECRETION OF INSULIN-LIKE GROWTH-FACTOR (IGF) IGF-II AND IGF BINDING PROTEIN-4 - ROLE OF IGF-II

The extent to which the insulin-like growth factor (IGF) system contri butes to the initiation and progression of colon cancer remains poorly defined. We recently reported that a majority of human colon cancers express and secrete the potent mitogen IGF-II and at least two inhibit ory binding proteins, IGFBP-2 and IGFBP-4. In the present study we mea sured the expression and secretion of IGF-II, IGFBP-2, and IGFBP-4 in relation to growth and differentiation of CaCo2 human colon cancer cel ls, which undergo spontaneous enterocytic differentiation in culture. Under the conditions of the present study, CaCo2 cells demonstrated an initial rapid phase of growth between Day 2 through Days 7-9 of cultu re, followed by a significant retardation in the growth between Days 9 -13. Alkaline phosphatase (ALP) activity, a marker of enterocytic diff erentiation, progressively increased between Days 7-13 in culture, tem porally correlating with post-confluent phase of negligible growth. Th ese changes in growth and differentiation were accompanied by >80% dec line in the relative concentration of IGF-II messenger RNA (mRNA) betw een Days 2-13. In contrast; the relative mRNA concentrations of inhibi tory binding proteins (IGFBP-2 and IGFBP-4) increased rapidly to 200% of Day 2 values by Days 5-7 before returning to baseline levels by Day 13. The relative protein concentrations of the three factors measured in the conditioned media of the cells followed a pattern very similar to that measured for the mRNA levels. While the changes in the relati ve protein concentrations and mRNA levels of IGF-II and IGFBP-4 were s tatistically significant, the changes measured in the RNA and protein levels of IGFBP-2 were not, as a result of large inter experimental va riations. Thus these results suggested that CaCo2 cell differentiation may require an attenuation of IGF-II effects. To confirm the latter p ossibility, additional studies were conducted with a specific neutrali zing antibody against IGF-II. Incubation of CaCo2 cells with anti-IGF- II antibodies from Day 0 through Day 7 significantly retarded the grow th of the cells and was accompanied by a significant increase in the c oncentration of alkaline phosphatase activity per 10(6) cells. Recentl y, we reported a potent inhibitory role of IGFBP-4 in the growth of co lon cancer cells. In the present studies, a possible important role of IGF-II is illustrated not only in the growth but also in the differen tiation of colonic cells. Our studies thus suggest that differential e xpression of IGF-II and IGFBPs may be playing a critical role in both proliferation and differentiation of colonocytes.