Production of insulin-like growth factor II (IGF-II) and different forms of IGF-binding proteins by HT-29 human colon carcinoma cell line

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Abstract
The serum‐free medium conditioned by the human colon cancer cell line HT‐29 contains insulin‐like growth factors (IGF) that are entirely complexed to binding proteins (IGF‐BP). Gel filtration in acid conditions of the cell‐conditioned medium permits separation of IGF‐BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF‐BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF‐I and IGF‐II radioreceptor assays, IGF‐I radioimmunoassay (RIA), and competitive protein‐binding assay specific for IGF‐II, it is shown that the IGF‐type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF‐II. Its concentration is ∼6 ng/106 HT‐29 cells with 60% present as a high‐molecular‐weight form of IGF‐II. This large 15 K IGF molecule is devoided of any IGF‐binding activity and might represent incomplete processing of pro‐IGF‐II peptide. By contrast, the level of IGF‐I detected by RIA is barely measurable and considered negligible (0.57 pg/106 HT‐29 cells). Although these IGF‐II‐like peptides exhibit a growth‐promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF‐I or IGF‐II, 3H‐thymidine incorporation into DNA of HT‐29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT‐29‐D4 clonal cells, derived from the parental HT‐29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.