Insulin binding to monolayer cell cultures of human fibroblasts, human colon carcinoma (HCT-8, HT-29), human breast carcinoma (MCF-7, T-47D), and melanoma (MM-96) was measured using 125I-insulin. Binding was time and temperature dependent in all cell lines, and only one cell line (MM-96) degraded 125I-insulin. High-affinity insulin-binding sites (Kd = 1.4 × 10-9m to 0.4 × 10-10m) were detected in all cell lines, and insulin-binding capacity ranged from 0.6 to 14 fmol/106 cells. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabeled insulin, dissociating surface-bound insulin and measuring residual receptors by 125I-insulin uptake. Exposure of tumor cells to >10-6m insulin for 2 hr at 37° led to a decrease in the number of insulin binding sites in MM-96 and colon cell lines only, with maximum down-regulation ranging from 58% (MM-96) to 88% (HCT-8) receptor loss. The decrease in insulin binding was due to a decreased number of receptors per cell with no change in affinity. Monolayers exposed to 1.7 × 10-5m unlabeled insulin for 7 hr at 37° invariably showed >50% receptor loss. However, monolayers exposed to 1.7 × 10-8m unlabeled insulin for 7 hr at 37° showed less marked (0 to 39%) down-regulation. In comparison, human fibroblasts showed 57% receptor loss after exposure to 3.5 × 10-9m unlabeled insulin for 7 hr. Thus, markedly supraphysiological concentrations of insulin are required to down-regulate insulin receptors in tumor cell lines compared with normal cells. This suggests a tumor-associated resistance to receptor down-regulation.

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This research was supported by the Medical Research Council of New Zealand.

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