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Glucocorticoid Receptor Determinations in Leukemia Patients Using Cytosol and Whole-Cell Assays¹

Laboratory of Molecular Endocrinology [S. I., V. N., P. L.], Departments of Histology [F. O. R.] and Pediatrics [R. M.], Sacro Cuore Catholic University; and Department of Hematology [G. D., D. P., F. M.], University of Rome, 00100 Rome, Italy

Parallel determinations of glucocorticoid receptors in the cells of patients with various forms of leukemia were made by two assay methods, one using cell-free cytosolic extracts and the other using whole-cell preparations. Both assays revealed saturable binding of triamcinolone acetonide in all cases. The mean equilibrium dissociation constant for the interaction of triamcinolone acetonide with the cytoplasmic receptor at 2° was 9.45 ± 6.33 (S.D.) nM while that for the whole-cell binding at 37° was 6.13 ± 3.25 nM, suggesting an increase in receptor affinity at physiological temperatures. Competition experiments with various unlabeled steroids revealed a higher degree of glucocorticoid specificity at 37° in whole-cell suspensions than at 2° in cytosol. In a comparative analysis of 41 leukemic cell specimens, it was found that determinations carried out by whole-cell assay, calculated as number of sites per cell, correlated well with those performed by cytosol assay, calculated as fmol/mg protein, independent of the type of leukemia. However, for cells with low receptor content, the two assay methods were more difficult to compare. In agreement with previous reports, the cytosol assay consistently underestimated the number of receptors with respect to the whole-cell assay, particularly in cases of lymphatic leukemia. Furthermore, the underestimation decreased for increasing levels of total cellular receptor. These results suggest that, in addition to possible defects in the cytoplasm-to-nucleus translocation process, the acceptor-binding capacity of the nucleus may also represent one of the factors which determines the levels of assayable cytoplasmic receptors. Moreover, they indicate that the two assay methods furnish nonequivalent information.

¹ Supported by Grant 800156696 of the Finalized Project "Control of Tumor Growth."

² To whom requests for reprints should be addressed.

Received 2/23/81. Accepted 6/15/81.