Insulin interaction with four human breast cancer cell lines in tissue culture was studied with respect to specific binding to receptors, degradation, and biological responsiveness. All four lines bound and degraded 125I-labeled insulin. Binding and degradation were time, temperature, and pH dependent. Unlabeled insulin competed with 125I-labeled insulin for binding to the breast cells with half-maximal inhibition of binding being observed with less than 0.6 ng/ml for three of the four lines. Other peptides competed for insulin binding in proportion to their biological potency. Scatchard analysis of the insulin binding data revealed curvilinear plots consistent with negative cooperativity, and this was confirmed by kinetic studies of dissociation. Quantitative analysis of insulin degradation revealed a similar Km for all four cell lines (1.0 to 2.2 × 10-7m), whereas maximal velocities varied over a 7-fold range. Bacitracin, a polypeptide antibiotic, inhibited insulin degradation by all four lines. In one cell line, typical competitive binding data and Scatchard plots were obtained only after inhibition of degradation with bacitracin. Insulin stimulated precursor incorporation into macromolecules and fatty acids in only two of the four cell lines; with these, significant stimulation was seen with concentrations as low as 0.6 ng/ml. No correlation was found between the amount of specific binding, receptor concentration, or receptor affinity and the ability of insulin to stimulate the cells, suggesting a defect distal to the hormone-receptor interaction in the two unresponsive lines. The two responsive cell lines showed the most insulin degradation. These human cell lines should provide a useful tool for further study of the complex mechanisms of insulin action and for the study of factors that regulate growth of human breast cancer.

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