HUMAN COLON-CARCINOMA CELLS (CACO-2) SYNTHESIZE IGF-II AND EXPRESS IGF-I RECEPTORS AND IGF-II M6P RECEPTORS/

The IGFs have been implicated in the development of the intestinal tra ct. We have studied the human colon carcinoma cell line CaCo-2 to gain more insight into the function of the IGFs in the gut. [I-125]IGF-I a nd -II bound specifically to CaCo-2 cells as measured in competitive b inding experiments. The existence of IGF-I receptors was further demon strated by affinity crosslinking studies using DSS as the crosslinking agent. Western blotting of CaCo-2 cell extracts using an anti IGF-II/ M6P receptor antiserum provided additional evidence for the expression of the IGF-II/M6P receptor. In addition, Northern blotting experiment s showed specific; IGF-I receptor and IGF-II/M6P receptor gene express ion in CaCo-2 cells. An 11 kb band was visualized with a 614 bp PstI I GF-I receptor probe on autoradiographs. Hybridization with a 663 bp IG F-II/M6P receptor probe yielded a 9 kb RNA species. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protection assays yielded two protected fragments, approximately 379 bases in length, with a 39 4 base IGF-I receptor riboprobe and a 250 base protected fragment with a 260 base IGF-II/MBP receptor riboprobe. In a subset of experiments a PstI 700 base fragment of the IGF-I cDNA and a 554 base SalI fragmen t of the IGF-II cDNA were used for hybridization: no hybridization was detected with the IGF-I probe. However, using the [P-32]IGF-II probe bands at 6.0 and 5.0 kb were labeled in Northern blotting experiments. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protec tion assays yielded a 289 base protected fragment and a faint 534 base species with a 556 base human IGF-II riboprobe. In addition, IGF-II i mmunoreactivity was measured in CaCo-2 cell-conditioned medium using a n IGF-binding protein blocked radioimmunoassay. CaCo-2 cell-conditione d medium contained 5-15 ng/ml IGF-II immunoreactivity. In conclusion, (1) CaCo-2 cells express both IGF-I receptor mRNA and IGF-II/M6P recep tor mRNA and contain functional IGF-I receptor and IGF-II/M6P receptor protein. (2) CaCo-2 cells express IGF-II mRNA and secrete IGF-II immu noreactivity. We hypothesize that in human colon carcinoma cells IGF-I I could act as an autocrine growth factor or alternatively could serve as a regulatory factor during differentiation.