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S S Simons

Publications and source records attributed to S S Simons.

89 records · Page 5Linked to original sources
PubMed1983

Dissociation of steroid binding to receptors and steroid induction of biological activity in a glucocorticoid-responsive cell.

Glucocorticoid responses in two independently derived lines of rat hepatoma tissue culture cells (HTC and FU5-5) were examined. FU5-5 cells exhibited induction of the enzyme tyrosine aminotransferase (TAT) at concentrations of dexamethasone that were approximately 7-fold lower than that required for HTC cells. FU5-5 cells also displayed substantial TAT induction with steroids that were partial agonists, or antagonists, in HTC cells. The increased sensitivity of FU5-5 cells was not, however, due to an increased affinity of FU5-5 cell receptors for dexamethasone, as determined from cell-free and whole cell binding experiments. The differential steroid sensitivity for TAT induction was observed with three other, structurally different glucocorticoids, thus apparently ruling out steroid metabolism in one of the cell lines as a cause. Also, induction of TAT in FU5-5 cells occurred at approximately 9-fold lower steroid concentrations than were required for the induction of glutamine synthetase (GS) in the same cells. Thus, the dose-response curves for TAT induction in HTC cells and for GS induction in FU5-5 cells are closely correlated with the saturation curve for whole cell steroid binding to receptor sites, while the dose-response curve for TAT induction in FU5-5 cells is shifted to lower steroid concentrations. This represents the first report of dissociation of two supposedly primary, glucocorticoid-induced functions and indicates that identical receptor-mediated processes cannot be utilized by FU5-5 cells for the induction of TAT and GS. The involvement of second messengers or different nuclear processes are possible explanations for the unusual behavior of FU5-5 cells during glucocorticoid induction of TAT.

Animals↗
PubMed1981

Dexamethasone 21-mesylate: an affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells.

We recently described the biological properties of an alpha-keto mesylate derivative of cortisol, cortisol-Mes. Cortisol-Mes exhibited long-term antiglucocorticoid activity, but there was no firm evidence that this activity was irreversible or receptor-mediated. Here we report that dexamethasone mesylate (Dex-Mes), which is the alpha-keto mesylate derivative of the more active glucocorticoid dexamethasone, is a candidate for a steroid-specific affinity label of glucocorticoid receptors. Dex-Mes is relatively stable, like cortisol-Mes, but possesses greater whole-cell antiglucocorticoid activity. However, Dex-Mes also possesses partial agonist activity, which is expressed at somewhat higher concentrations of Dex-Mes than the antagonist activity. Dex-Mes is more efficient than cortisol-Mes in competing for dexamethasone binding to glucocorticoid receptors. Furthermore, Dex-Mes is effective at lower concentrations than cortisol-Mes in causing long-term apparently irreversible antiglucocorticoid effects in whole and broken cells. The cell-free effect of Dex-Mes is specifically prevented by coincubation with an excess of cortisol. These facts argue that the apparently irreversible effects of Dex-Mes are steroid mediated. [3H]Dex-Mes has been used to identify a glucocorticoid-specific, covalently labeled fraction on sodium dodecyl sulfate/polyacrylamide gels with a molecular weight of approximately 85,000. Thus Dex-Mes appears to have been established as an affinity label for glucocorticoid receptors.

Affinity Labels↗
PubMed1980

Unique long-acting antiglucocorticoid in whole and broken cell systems.

The biological properties of cortisol 21-mesylate (CM), an alkylating derivative of cortisol, were investigated in a line of rat hepatoma tissue culture (HTC) cells. CM appears to bind to glucocoticoid receptors in cell-free extracts because CM inhibits the specific binding of [3H]dexamethasone. However, in whole cells CM not only fails to induce the enzyme tyrosine aminotransferase (TyrATase) but also inhibits the induction of TyrATase by dexamethasone. Thus CM is an antiglucocorticoid. This is not caused by cell death, because CM is relatively nontoxic up to concentrations of 10 microM. The concentration of CM needed for half maximal inhibition of TyrATase induction is an order of magnitude lower than that predicted from the apparent cell-free affinity of CM for the glucocorticoid receptors of HTC cells, which suggests that the cell-free binding data does not reflect an equilibrium situation. In fact, the reactive alpha-keto mesylate group was intentionally incorporated into cortisol in hopes of obtaining a steroid capable of undergoing irreversible reactions. When HTC cells were preincubated with either CM or the reversible antiglucocorticoid progesterone and then washed to remove free steroid, only the CM-treated cells failed to show subsequent induction of TyrATase by dexamethasone. Furthermore, preincubation of HTC-cell cytosol with CM blocked approximately 75% of the subsequent exchange binding of [3H]dexamethasone to glucocorticoid receptor sites. Thus, the actions of CM in whole and broken cells either require an exceptionally long time for reversal or are not reversible. Together, these results indicate that CM is a unique antagonist and could be an irreversible antiglucocorticoid in vitro.

Animals↗
PubMed1979

Factors influencing association of glucocorticoid receptor-steroid complexes with nuclei, chromatin, and DNA: interpretation of binding data.

Attempts to reconstruct, in a test tube, the steroid-hormone system of a responsive cell are fraught with enumerable difficulties. In this chapter I have attempted to point out some of the factors that affect receptor-steroid complexes and their interactions with acceptors. In most cases there is a quantitative influence of these factors on the level of steroid complex binding to acceptors. In some cases, selected experimental designs that neglect these factors and methods of presenting the observed data may lead to artifactual conclusions. Several of these problems should disappear when the prospect of pure receptor-steroid complexes [127, 147, 150, 181, 247, 248] becomes a common occurrence. Nevertheless much has already been learned about the interactions of complexes with acceptors, which in turn have been used to help formulate models of steroid-hormone action.

Animals↗
PubMed1977

Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells.

Binding to DNA associated with cellulose has been used to investigate the receptor-glucocorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binging form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.

Carcinoma, Hepatocellular↗
PubMed1977

Glucocorticoid receptor-steroid complex binding to DNA. Competition between DNA and DNA-cellulose.

The binding of the glucocorticoid receptor-steroid complex from a line of rat hepatoma tissue culture (HTC) cells to DNA has been examined. An equilibrium competition assay involving a constant, low total amount of double-stranded DNA was developed to compare the complex binding ability of DNA free in solution and bound to cellulose. This binding ability is lowered by a factor of five when DNA is associated with cellulose. Similar studies with HTC cell, calf-thymus, and Escherichia coli DNA revealed no difference in the relative number or affinity of binding sites for receptor-steroid complex in each DNA. The synthetic DNA molecules poly[d(A-T)-d(A-T)] and poly[d(G-C)-d(G-C)] bound complexes equally well but less than the three "natural" DNA molecules. This appears to be due to differences in acceptor site affinity and suggests that nucleotide complexity and/or sequence influences the affinity of HTC cell receptor-glucocorticoid complexes for DNA.

Binding, Competitive↗
PubMed1976

Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.

Binding Sites↗