The karyotype of the glucocorticoid-sensitive, lymphoblastic human T-cell line CCRF-CEM shows a unique deleted and inverted chromosome 9.

Chromosome karyotypes were prepared from the highly glucocorticoid-sensitive clone C7 of the human acute lymphoblastic leukemia T-cell line CCRF-CEM. The modal number of chromosomes is 47, and one chromosome #9 has a pericentric inversion of the heterochromatin region (9qh) plus a deletion of the short arm. In most cells, there is an extra chromosome #20. All other chromosomes appear to be normal. Examination of the uncloned line CCRF-CEM (ATCC CCL 119), which was frozen away shortly after the line was originated and has undergone fewer passages than CEM C7, also revealed the same abnormality of chromosome #9. Each of 10 other clones isolated from CCRF-CEM in this laboratory also contained the abnormal chromosome #9.

Glucocorticoid-resistant human acute lymphoblastic leukemic cell line with functional receptor.

A receptor-containing, steroid-resistant clone of CEM cells, CEM-C1, was isolated without selective pressure from the wild-type population. The biological and physicochemical properties of glucocorticoid receptors in CEM-C1 cells were compared to those from a clone (CEM-C7) sensitive to glucocorticoid-mediated lysis. In a whole-cell binding assay, CEM-C1 cells exhibited high affinity for [3H]dexamethasone (Kd, 22 nM), nuclear translocation of steroid:receptor complex (nt, 43%) and were found to contain, on the average, 12,000 receptor sites/cell (R0). These steroid-binding parameters were similar to those displayed by wild-type CEM-C7 cells: Kd, 19 nM; nt, 47%; and R0, approximately 14,000 sites/cell. The ion-exchange and gel permeation profiles were indistinguishable from those of identically treated CEM-C7 cytosols. Thus, diethylaminoethyl cellulose chromatography of CEM-C1 cytosol showed that [3H]triamcinolone acetonide:receptor complex was eluted at 50 mM phosphate and 220 mM phosphate under "activating" and "nonactivating" conditions, respectively. Receptor complex of activated CEM-C1 cytosol bound to DNA-cellulose and was eluted at 100 mM salt. Filtration of unactivated CEM-C1 cytosol over Sephacryl S-300 generated a single peak of radioactivity for receptor complex with a calculated Stokes' radius of 55 to 59 A. Dexamethasone induced glutamine synthetase in CEM-C1. The dose dependence (50% effective dose, approximately 20 nM) and maximal fold increase (1.9, 1 microM dexamethasone) were comparable to those observed in CEM-C7. Since CEM-C1 cells contain apparently normal, functional cytosolic receptor, the results suggest that resistance to glucocorticoid in these cells involves a defect(s) at another locus.

Lymphocytolytic activity of glucocorticoids in vitro: 51Cr release under equilibrium conditions.

Cytolytic activity of glucocorticoids in vitro is assessed by measuring radiochromium release from steroid-treated thymic lymphocytes under the equilibrium conditions provided by a continuous-labeling technique. Isotope release is a glucocorticoid-specific effect produced at physiological concentrations and is virtually abolished by inhibitors of RNA and protein synthesis. The relative lytic potencies of the steroids tested are comparable to those reported for glucocorticoids as measured by other methods. This procedure not only possesses the advantages typical of isotopic techniques in general, but, in addition, circumvents the problem of "spontaneous" label release associated with the pulse-labeling method. It is a useful alternative to the morphologic examination of cells or the estimation of cell viability for determination of glucocorticoid cytolytic activity.

Spontaneous 51Cr release by isolated rat hepatocytes: an indicator of membrane damage.

Radiochromium uptake and release by isolated rat hepatocytes in suspension was monitored under continuous-labeling conditions. Cell protein remained unchanged during the absorption phase, whereas the release of 51Cr correlated well with the loss of cell viability and release of cytoplasmic protein. The results suggest that under equilibrium conditions, 51Cr release represents an efflux of label from damaged or dying preparations and not an elution of radioisotope from intact cells.