Sucrose Density Gradient Analysis of 1, 25-Dihydroxyvitamin D₃ Binding in Human Breast Tumors

The object of this investigation was to detect the type and number of 1, 25-dihydroxyvitamin D₃ [1, 25-(OH)2D3]-binding proteins present in breast tumor tissue and thus to assess, by using sucrose density gradient analysis, the significance of the presence of 1, 25-(OH)₂D₃ binding in breast tumors. Cytosol fractions from 25 tumors (24 malignant breast tumors and 1 benign breast mass) were analyzed for the binding of vitamin D3 metabolites by sucrose density gradient ultracentrifugation. Cytosol fractions from 13 breast tumors exhibited binding of 1, 25-(OH)₂D₃ to a 4.1S macromolecule. Only 5 of the tumors (20%) showed a specific binding protein for 1, 25-(OH)₂D₃, which had a sedimentation coefficient of 3.2-3.4S, similar to that of the 1, 25-(OH)₂D₃ receptor protein found in intestinal cytosol. However, levels below 8 fmol/mg protein were observed for the positive malignant tumors compared to receptor concentrations of 286-563 fmol/mg protein which have been reported for the adult mammalian intestine. No correlation was found between the presence or absence of a specific 3.2-3.4S binding protein and the levels of estrogen receptors. Two of the breast tumors showed binding of 1, 25-(OH)₂D₃ to the 5-6S 25-hydroxyvitamin D₃-binding protein. Cytosol, fractionated by precipitation with ammonium sulfate at 40% saturation in order to reduce interference from either the 5-6S binding protein or the 4.1S complex, showed no specific binding of 1, 25-(OH)₂D₃. Rachitic chick intestinal cytosol, vitamin D-deficient rat intestinal cytosol, and cytosol from MCF 7 cells, used as positive controls, consistently exhibited the characteristic 3.2-3.6S receptor. We conclude, due to a heterogeneous cellular population and possible serum contamination in human breast tumors, that the characterization of 1, 25-(OH)₂D₃ receptors in breast cancer cells compared to normal cells may provide more definitive conclusions concerning the relationship between 1, 25-(OH)₂D₃ receptors and malignancy.