Immunocytochemical application of monoclonal antibodies to rat liver glucocorticoid receptor.

Seven per cent (10/145) of hybridomas raised against partially purified activated glucocorticoid receptor from rat liver produced monoclonal antibody to receptor. Six IgM secreting clones selected for further investigation bound equally well to activated and non-activated receptor from fresh rat liver, but significantly less well (11-25 per cent) to receptor from frozen rat liver. No interaction was found with oestrogen receptor from rat uterus but extensive cross reaction occurred with progesterone receptor. Although none of the antibodies bound to glucocorticoid receptor from human or porcine liver or lymphoid cells, several cross-reacted with mouse liver glucocorticoid receptor. Immunoelectroblotting of proteins from fresh and frozen rat liver cytosol showed the antibodies bound to 90,000 and 40,000 MW forms of receptor respectively. Immunostaining of both frozen and paraffin embedded sections of rat tissue showed that receptor is preserved during fixation and processing of tissues. Using both indirect immunoperoxidase and immunogold silver staining methods, the pattern of receptor staining observed correlates with the known glucocorticoid responsiveness of the tissues studied.

Glucocorticoid binding and cytolethal responsiveness of hairy-cell and chronic lymphocytic leukaemia.

The glucocorticoid binding properties and cytolethal responsiveness of leukaemic cells were studied in vitro in seven patients with hairy-cell leukaemia (HCL) and five with chronic lymphocytic leukaemia (CLL). Substantial levels of glucocorticoid binding were detected both in whole cell and cytosol preparations from all patients although the level of binding by HCL cells always exceeded that of CLL cells (P less than 0.05). In both leukaemic cell types the uptake and binding of prednisolone in vitro was significantly greater than that of dexamethasone (P less than 0.05). CLL cells showed a variable dose-related cytolethal response to methylprednisolone sodium succinate (MPSS) treatment in vitro although cytolytic effects were not marked in the usual pharmacological dose range (10(-5)-10(-6)M). Treatment of CLL patients with conventional doses of prednisone for extended periods or high intravenous infusions of MPSS over shorter periods had no consistent effect on the in-vitro level of steroid binding or the cytolethal responsiveness of CLL cells to glucocorticoid treatment. Although HCL cells proved highly resistant to the cytolethal effects of MPSS in vitro, the substantial binding of glucocorticoids by leukaemic cells from all HCL patients indicates the potential value of steroid therapy in this disease should be explored further.

Human lymphoid cell lines and glucocorticoids: II. Whole cell and cytoplasmic binding properties of lymphoblastoid, leukaemia and lymphoma lines.

The glucocorticoid binding properties of 18 human lymphoid cell lines (HLCL) have been investigated. The specificity of steroid binding was confirmed with various glucocorticoid agonists and antagonists. A gradation in whole cell and cytoplasmic glucocorticoid binding capacity was observed in the different cell line types: lymphoblastoid greater than lymphoma greater than leukaemia. The cytoplasmic receptors of leukaemia and lymphoblastoid lines appeared to contain both proteinaceous and phospholipid components. Cytoplasmic steroid-receptor complexes exhibited a wide range of sedimentation coefficients (8.5-11.3S) in low ionic strength buffer but there was no correlation with cell line type or glucocorticoid sensitivity. Activation of these complexes by heat (37 degrees C) or exposure to high ionic strength buffer (0.3 M NaCl) induced nuclear binding of steroid but only complexes in high ionic strength buffer manifested changes in sedimentation coefficient. No correlation was observed between the level or nature of glucocorticoid binding and the cytolethal or cytostatic responsiveness of HLCL to glucocorticoid treatment in vitro. The resistance to cytolethal effects cannot be ascribed to a failure of cells to take up and bind steroid or to significant differences in the molecular species of cytoplasmic receptors present. The molecular mechanisms by which glucocorticoids achieve cytolethal responses in human lymphoid cells has still to be resolved.

Human lymphoid cell lines and glucocorticoids: I. Characterization and cytolethal responses of lymphoblastoid, leukaemia and lymphoma lines.

Using various genotypic and phenotypic markers 20 human lymphoid cell lines have been classified as lymphoblastoid, leukaemia or lymphoma subtypes. Each cell line type exhibited characteristic morphological and behavioural properties in suspension culture. Whilst most lymphoblastoid and lymphoma cell lines manifested B-cell phenotypes and contained Epstein Barr virus (EBV) genome, leukaemia lines demonstrated T-cell markers and lacked EBV genome. Individual cell lines demonstrated unique isoenzyme profiles for the seven polymorphic enzymes studied without subtype specificity. None of the cell lines studied was entirely homogeneous although lymphoblastoid lines contained only a minor subpopulation of other cell line types. The mixed population of cells indicate the need for caution in the use of these cell lines as in vitro models of lymphoid cancer and suggests further refinement of classification methods is required. Incubation of different cell line types with prednisolone for 48-168 h revealed most were highly resistant to cytolethal and cytostatic effects of glucocorticoids in vitro. Suprapharmacological doses of steroid (10(-3) M) were required in most instances before significant cytolethal responses occurred. Only one lymphoblastoid, one lymphoma and two leukaemia lines responded to pharmacological doses (10(-5)-10(-6) M) of prednisolone.