HUMAN COLON CARCINOMA CELLS (CACO2) EXPRESS IGF RECEPTORS AND SYNTHESIZE IGF-II: POTENTIAL AUTOCRINE OR PARACRINE ACTION OF IGF-II

Abstract

The IGFs have been implicated in the development of the intestinal tract. We have studied the human colon carcinoma cell line CaCo2 to gain more insight into the function of the IGFs in the gut.¹²⁵I-IGF-I and -II bound specifically to CaCo2 cells as measured in competitive binding experiments. The existence of IGF-I receptors and of IGF-II/M6P receptors was further demonstrated by affinity crosslinking studies using DSS as the crosslinking agent. Western blotting of CaCo2 cell extracts using an anti IGF-II/M6P receptor antiserum provided additional evidence for the expression of the IGF-II/M6P receptor. Northern blotting experiments showed specific IGF-I receptor and IGF-II/M6I receptor gene expression in CaCo2 cells: cells were homogenized in 4M guanidiniumthiocyanate and RNA extracted in 5.7 M CsCI. RNA was denatured in glyoxal/DMSO and electrophoresed on 0.8 % agarose. RNA was transferred to a nylon membrane by capillary transfer, fixed and the blots hybridized with cDNA probes. A 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp fragment of the IGF-II/M6P receptor cDNA were labeled with ³²P-dCTP using random prime labeling: Autoradiographs of Northern blots showed a 11 kb band with the IGF-I receptor probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species. In a subset of experiments a Pst I 700 bp fragment of the IGF-I cDNA and a 833 bp Pst I fragment of the IGF-II cDNA, were used for hybridization: no hybridization was detected with the IGF-I probe. However, using the ³²p.IGF-II probe bands at 6.4, 5.3 and 5.0 kb were labeled. In addition, CaCo2 cells were cultured until confluency, changed serum-free and cell-conditioned medium was harvested. IGF-II immunoreactivity was measured using an IGF-binding protein blocked radioimmunoassay. CaCo2 cell-conditioned medium contained 1-2 ng/ml IGF-II immunoreactivity. In conclusion. (1) CaCo2 cells express both IGF-I and IGF-II/M6P receptors. (2) CaCo2 cells express IGF-II mRNA and secrete IGF-II immunoreactivity. We hypothesize that in human colon carcinoma cells IGF-II acts as an autocrine growth factor

(Supported by DFG and DAAD. Bonn, Germany).

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