Richard J. Milholland

Cortexolone: Binding to glucocorticoid receptors in rat thymocytes and mechanism of its antiglucocorticoid action

Abstract In rat thymocytes, cortexolone (11-deoxycortisol) competes for binding to glucocorticoid receptors, as identified on sucrose density gradients, and blocks the effect of triamcinolone acetonide (TA) on 2-deoxyglucose uptake. The cellular distribution and sedimentation coefficients of 3H-TA-receptor complexes are dependent on the incubation temperature and ionic strength of the extraction buffer. In contrast, the single receptor complex formed by 3H-cortexolone has a sedimentation coefficient of 3.5S, and is unaffected by changes in temperature and salt concentration.

Glucocorticoid activity of various progesterone analogs: correlation between specific binding in thymus and liver and biologic activity.

When tested in an in vitro assay system, progesterone and various analogs of this steroid were shown to compete with [3H] triamcinolone acetonide (TA) for specific glucocorticoid receptors in both rat liver and thymus. Of these analogs, the following derivatives of progesterone were potent competitors of TA binding and, when injected into adrenalectomized rats, induced regression of the thymus and marked increases in hepatic tyrosine aminotransferase activity: 11 beta-hydroxyl, 6 alpha-methyl, 6 alpha, 16 alpha-dimethyl, and 6 alpha-methyl-17 alpha-hydroxyl. In contrast, progesterone, 16 alpha-methyl, and 17 alpha-hydroxy progesterone competed with TA in vitro but failed to elicit either gluco- or antiglucocorticoid activity in vivo. Also, we observed that the oral contraceptive 6 alpha-methyl-17-(1-propynyl)testosterone competes very effectively with TA in a cell-free preparation of rat liver and induces an increase in hepatic tyrosine aminotransferase activity. The 11 beta-hydroxyl group has previously been thought to be essential for glucocorticoid activity. Our studies indicate that substitution of progesterone or testosterone with a 6 alpha-methyl group negates the need for an 11 beta-hydroxyl substitutuent as a prerequisite for glucocorticoid activity.

Studies on the antiglucocorticoid action of progesterone in rat thymocytes: Early in vitro effects☆

Abstract Progesterone was found to exhibit antiglucocorticoid properties in rat thymocytes, as indicated by its ability to compete effectively for triamcinolone acetonide (TA) binding sites as well as to inhibit the biochemical effects of TA. In a cell free system, progesterone competitively inhibited the binding of TA to glucocorticoid-specific binding sites and was more effective than the antiglucocorticoid cortexolone in this respect. Progesterone at 10−7−10−6M antagonized the inhibitory effect of 5 × 10−8TA on RNA metabolism, although it was inactive by itself as an inhibitor of [3H]-uricline uptake and incorporation into thymocyte RNA; this antagonism was not due to competition by progesterone for TA uptake into the cells. Progesterone was less efficient than cortisol or TA in depleting the cytosol receptors. However, unlike the glucocorticoid agonists which formed a temperature-dependent, salt extractable nuclear bound complex, progesterone was bound tightly to a fraction in the 27,000 g pellet which was unaffected by changes in the incubation temperature or salt extraction conditions. Additional binding of progesterone was observed in cells pre-saturated with TA. suggesting the presence in rat thymocytes of progesterone binding components other than the glucocorticoid receptors.

A comparison of the effect of glucocorticoids on glucose uptake and hexokinase activity in lymphosarcoma p1798.

Abstract Exposure of cortisol-sensitive lymphosarcoma P1798 cells to 1 · 10 −6 M dexamethasone for 3 h resulted in a marked depression in the uptake of labeled 2-deoxyglucose. The majority of the trichloroacetic acid-soluble radioactivity was present as deoxyglucose 6-phosphate, with a small amount present as the free sugar. Approximately a 60% reduction was seen in both intracellular deoxyglucose and deoxyglucose 6-phosphate following exposure to the hormone. The inhibitory effect of glucocorticoids on glucose uptake was compared with their effect on tumor hexokinase activity after treatment both in vivo and in vitro . No change in tumor hexokinase activity could be detected at times when there was a significant inhibition of glucose uptake. No detectable glucokinase activity was found in untreated tumors. From these experiments it may be concluded that glucocorticoids are acting to decrease the entry of glucose into sensitive P1798 cells. Several possible modes of corticosteroid action on glucose uptake are discussed.

Mechanism of Action of Glucocorticoids

Although recent advances in our knowledge of hormone action have been impressive, there is at present no clear understanding of how any hormone acts at the molecular level. This is one of the major unsolved problems in biology today. It seems evident that one or more hormones are involved in the growth and metabolism of most normal tissues. The fact that certain tumors are hormone dependent or responsive also indicates that hormones may play an important role in the etiology and treatment of cancer. There is little doubt that knowledge concerning hormone action is likely to yield new insight and provide different approaches to the treatment of various diseases, including cancer.

In vivo antiglucocorticoids: Comparison between in vivo activity and in vitro competition of progestins for the glucocorticoid receptor

Abstract The in vivo antiglucocorticoid activity of representative progestins was compared with in vitro competition for dexamethasone binding to the liver glucocorticoid receptor. In vivo antagonism of dexamethasone (5 μg/rat) induction of glycogen deposition, tryptophan oxygenase activity, and tyrosine amino transferase activity was determined at several concentrations of the steroids. Antagonism of glycogen deposition and tryptophan oxygenase activity was readily demonstrated in both intact and adrenalectomized rats. Tyrosine aminotransferase activity, however, was not antagonized with any of the test steroids at any time point. All steroids tested displaced at least 40% of the dexamethasone bound to the liver receptor in vitro but no clear relationship between in vivo and in vitro activity was obvious. These results raise the issue of: (1) the usefulness of competition studies for evaluating potential antiglucocorticoids, and (2) the possibility of differential in vivo antagonism of glucocorticoid responses.

Evidence for a factor in liver cytosol which controls the competition between progesterone and triamcinolone acetonide for the glucocorticoid receptor

Abstract There is an inhibitory factor in rat liver which prevents progesterone and certain of its analogues from competing with triamcinolone acetonide for binding to the blucocorticoid receptor. The effects of this inhibitor can be eliminated by dilution of the cytosol or by gel filtration. Neither rat thymus nor serum contain this factor.

The effect of hydrocortisone treatment on the in vivo phosphorylation of a subgroup of non-histone nuclear proteins in the mouse lymphosarcoma P1798.

Abstract The in vivo phosphorylation of a small subgroup of non-histone nuclear proteins, similar to the high mobility group (HMG) proteins described by Goodwin and Johns ( Methods in Cell Biology (1977) Vol. XVI, eds. G. Stein, J. Stein and L.J. Kleinsmith, pp. 257–267, Academic Press, New York), was studied in the P1798 mouse lymphosarcoma. A single injection of cortisol, resulting in a 50% reduction in tumor mass of the sensitive strain of this tumor, caused a marked suppression in phosphorylation of at least three of these proteins, independent of any apparent change in protein concentration. No such change was observed in the cortisol-resistant strain, and it is suggested that the effect of cortisol in causing regression of this tumor might be mediated via changes in phosphorylation of specific nuclear proteins.

Binding of the glucocorticoid receptor complex to the nucleosomal core in the P1798 mouse lymphosarcoma.

Binding of the glucocorticoid receptor complex to nucleosomes has been studied using the mouse P1798 lymphosarcoma. Cells were incubated with [3H]triamcinolone acetonide (TA), and nuclei prepared and digested with 3 different concentrations of micrococcal nuclease. After fractionation with EDTA and NaCl, it was observed that [3H]TA bound with similar specific radioactivity to mononucleosomes containing both core and linker DNA, of 183 +/- 5, and 168 +/- 4 base pair lengths, respectively, as well as to core size DNA, of 148 +/- 3 base pair length, suggesting that the glucocorticoid receptor bound to the core portion of the nucleosome. Steroid binding was found to be associated with regions of the nucleosome that were depleted in histone H1 and enriched in high mobility group (HMG) proteins 1 and 2; only negligible binding was noted in nucleosomes enriched in histone H1 and depleted in HMG proteins. In addition to binding to core nucleosomes, the glucocorticoid receptor complex was also shown to bind to a fraction sedimenting at 5-6 S on sucrose gradients characterized by subnucleosome and mononucleosome size DNA, as well as by core histones. While binding of the steroid receptor complex to linker regions of the nucleosome cannot be ruled out, this data would appear to present the first concrete evidence that glucocorticoid binding, at least in the P1798 lymphosarcoma, is to core nucleosomes. Some caution in interpretation of the results is indicated, however, on 2 points: (1) receptor redistribution during nuclease digestion cannot be ruled out; (2) only the binding of a small proportion of the steroid receptor complex may be physiologically relevant.