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.

Cytolethal sensitivity of human lymphoid cells to glucocorticoids and oxidised polyamines.

A series of clonally-derived glucocorticoid-sensitive and -resistant human lymphoid cell lines was used to investigate the relationship between sensitivity to the effects of oxidised polyamines and initiation of glucocorticoid-induced cytostatic and cytolethal responses. Whilst the exogenous polyamines were found to exert no effect by themselves, incubation of cells for 48 h with 10(-4)M spermine or spermidine in the presence of serum polyamine oxidase produced severe lethal responses in all clones tested. By contrast 10(-5)M exogenous polyamines in the presence of polyamine oxidase produced lethal effects only in glucocorticoid-sensitive clones. Spermine was more potent than spermidine. The significance of these observations is discussed.

Mode of cell death induced in human lymphoid cells by high and low doses of glucocorticoid.

The kinetics, specificity and morphology of cytolethal responses have been studied in human glucocorticoid-sensitive and -insensitive lymphoid cell lines (HLCL) and fibroblasts following treatment with high (10(-3)M) and low (10(-6)M) doses of steroid. The high dose cytolethal response appears non-specific occurring in all cell lines with every steroid tested. By contrast, the low dose (pharmacological) cytolethal response requires an active glucocorticoid and a sensitive HLCL. However, both high and low concentrations of steroid induce virtually identical morphological changes in dying cells and similar changes can be induced in cells killed by deliberate feed exhaustion. Although the morphological features in each case resemble apoptosis, the "programmed" physiological form of cell death, the intracellular events leading to cytolysis seem likely to differ. The earliest morphological changes presaging cell death comprise rounding up of cells and condensation of nuclear chromatin. Nuclear changes progress rapidly thereafter and appear to result from detachment of chromatin from the nuclear matrix. The low dose cytolethal response requires the continuous presence of glucocorticoid for periods in excess of 24h, prior to which cell growth appears unaffected. The constancy of this latent interval suggests glucocorticoids may influence some replication control mechanism unrelated initially to macromolecular biosynthesis.

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.