Ethanol calories do not enhance breast cancer in isocalorically fed C3H/Ou mice.

Mammary tumorigenesis is augmented when C3H/Ou mice are fed diet ad libitum but delayed when calories are restricted by 40%. Three feeding experiments were done to evaluate the effect of ethanol on mammary tumorigenesis in isocalorically fed C3H/Ou mice: 1) ad libitum feeding of semipurified solid diet, with one group receiving 12% ethanol (15 g/kg/day) in the drinking water while controls received water alone; 2) isocaloric pair feeding of semipurified solid diet, with ethanol (4 g/kg/day) administered by gavage five time per week; and 3) isocaloric pair feeding of Lieber-DeCarli liquid diet, with one group receiving 29% of calories as ethanol (20 g/kg/day) in the diet. Despite administration of ethanol to isocalorically fed C3H/Ou mice for 65 weeks by three different methods, mammary tumor development was not enhanced. In two of the three ethanol-consuming groups, weight gain and mean body weight were less in the ethanol-consuming mice than in the controls, despite equal total calorie consumption. In only one ethanol-consuming group, where mice received ethanol as a 12% solution in the drinking water, was any difference noted in the tendency to develop mammary tumors. In this case, delay in tumorigenesis was apparent in the ethanol-consuming animals (p = 0.03). These findings do not support the hypothesis that ethanol calories augment the risk of breast tumorigenesis among breast cancer-prone mice consuming isocaloric diets. Instead, reductions in weight gain and body weight among ethanol-consuming mice and an apparent reduction in mammary tumorigenesis in one of three experimental groups suggest that ethanol may decrease metabolic utilization of calories and hence contribute to lowered energy availability.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells.

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.

Ketoconazole inhibition and glucocorticoid action in the human lymphoblastic leukemia cell line CEM-C7.

The antifungal drug, ketoconazole, was reported to antagonize the induction of the enzyme tyrosine aminotransferase (TAT) by glucocorticoids in hepatoma tissue culture (HTC) cells, and to compete with glucocorticoids for binding to the glucocorticoid receptor. Since glucocorticoids inhibit the growth of the human leukemia cell line CEM-C7, ketoconazole might be expected to reverse this inhibition. Unexpectedly, ketoconazole inhibited CEM-C7 cell growth without utilizing glucocorticoid receptors. This was confirmed by ketoconazole inhibition of the growth of a receptor-less subline of CEM-C7 cells which are insensitive to glucocorticoids. Ketoconazole competed with triamcinolone acetonide (TA) for binding to the glucocorticoid receptor in cell-free supernatant prepared from CEM-C7 cells, but this was greatly reduced if ketoconazole and TA were incubated with intact cells prior to preparation of the cell-free supernatant. Ketoconazole inhibited induction by TA of the enzyme glutamine synthetase only at concentrations of 45-90 microM. We conclude that ketoconazole antagonism of glucocorticoid activity in CEM-C7 cells is probably not of pharmacologic significance due to the large concentrations required, and its reduced interaction with receptors in intact cells. The growth inhibitory activity of ketoconazole may be of interest in cancer chemotherapy.

Glucocorticoid receptor properties and glucocorticoid regulation of glutamine synthetase activity in sensitive C6 and resistant C6H glial cells.

The relationship between induction of glutamine synthetase activity by dexamethasone and binding of the steroid to cytosolic glucocorticoid receptors was examined in sensitive C6 and resistant C6H glial cell cultures. Glutamine synthetase activity increased 3-4-fold when C6 cultures were exposed to 7.6 x 10(-6) M dexamethasone. This inductive response was reversible, dose-dependent (ED50 approximately 2 x 10(-8) M), required de novo protein and RNA synthesis, and was elicited only by glucocorticoid steroids. Progesterone, but not epicortisol, antagonized the dexamethasone-induced enzyme increase. In contrast, only a slight inductive effect was observed in dexamethasone-treated C6H cells. Competitive binding assays demonstrated that specific binding of [3H]-dexamethasone to cytosolic receptors was also dose-dependent. The ED50 was approximately 10(-8) M for both C6 and C6H cells. Scatchard analysis revealed that each C6 cell contained approximately 10,800 receptor sites and that the equilibrium dissociation constant (Kd) was 4.5 x 10(-9) M. Each C6H cell possessed approximately 12,200 sites, and the Kd was 6.7 x 10(-9) M. Unlabeled dexamethasone and cortisol (but not epicortisol) competed effectively with [3H]-dexamethasone for binding to cytosolic receptor sites and nuclear sites of both cell types. These results suggest that induction of glutamine synthetase activity in dexamethasone-treated C6 cells is a glucocorticoid-directed response. Since C6H cells are refractory in this regard but contain functional cytosolic receptors which interact with cell nuclei, the basis for their resistance appears to involve some step beyond these cellular processes.

Response of isolated guinea-pig tracheal muscle to glucocorticoid and non-glucocorticoid succinates.

Glucocorticoids have been reported to relax tracheal smooth muscle and to potentiate its response to isoproterenol. For this reason, isolated tracheal muscle has been studied as a model system for exploring some of the mechanisms of glucocorticoid action in the therapy of asthma. Various glucocorticoid succinates and phosphates were tested in guinea-pig tracheal muscle for relaxing activity ahd potentiation of the isoproterenol response. In both of these activities, the glucocorticoid succinates were more effective than the corresponding phosphates. There were no differences in potency among the glucocorticoids commensurate with their antiasthmatic activity. In comparing the succinate derivatives of glucocorticoids with non-glucocortiocod succinates, it was found that while 10(-3) M glucocorticoid succinate was required to demonstrate relaxation, the non-glucocorticoids were capable of producing complete relaxation at 10(-3) M and demonstrable relaxation at 10(-4) M. The latter were also capable of potentiating the isoproterenol response at a dose of 10(-4) M. It apears that these responses to glucocorticoids are due to the succinate derivative form of the steroid and not to the glucocorticoid itself. This wound suggest that glucocorticoid succinate relaxation and its isoproterenol potentiation in tracheal muscle does not provide an apropriate model for studying the mechanisms of the antiasthmatic effect of glucocorticoids.

A glucocorticoid receptor in fetal mouse: its relationship to cleft palate formation.

Fetal mouse tissue was investigated for a glucocorticoid binding receptor which might be responsible for cleft palate formation. Fetal mouse heads contain a soluble component which binds the glucocorticoid triamcinolone acetonide in vitro with high affinity. This binding component is present in small finite amounts. Other glucocorticoids compete with triamcinolone acetonide for the binding site in a manner consistent with their potency ranking as cleft palate teratogens. Several mineralocorticoids and progestins also compete when administered in vitro but not when administered in vivo. Triamcinolone acetonide binding was determined in three mouse strains, A/J, C3H, and C57BL, which are listed in decreasing order of cleft palate susceptibility to cortisone. No positive correlation was found between cortisone cleft palate susceptibility and either triamcinolone acetonide binding affinity or binding amount in fetuses from these strains. Cleft palate dose response curves for triamcinolone acetonide were determined in these strains, but they were not parallel to each other as they were for cortisone. This suggests that triamcinolone acetonide may cause cleft palate by different mechanisms in these strains. Thus, fetal mouse tissue contains an apparent glucocorticoid receptors, but its relationship to cleft palate formation in mice is not clear.

A low-molecular-weight protein from rat liver that resembles ligandin in its binding properties.

A protein of S20,W 1.6S and mol.wt. 14000, which binds covalently a metabolite of the aminoazodye carcinogen NN-dimethyl-4-amino-3'-methylazobenzene, was isolated from rat liver cytosol from both carcinogen-treated and normal rats. The protein binds non-covalently palmitoyl-CoA, fatty acids, bilirubin, sex steroids and their sulphates, bile acids and salts, bromosulphophthalein, diethylstilboestrol and 20-methylcholanthrene with a wide range of affinities. The protein is isolated as three components with isoelectric points of 5.0, 5.9 and 7.6 by a method involving isoelectric focusing. All three components have closely similar amino acid analyses, tryptic-peptide 'maps' and u.v. spectra. Each single component redistributes into all three on further electrophoresis. However, the three forms differ in their binding characteristics, the form of pI 7.6 having much the highest affinity for compounds bound non-covalently. The protein was identified immunologically in rat liver, small intestine, adipose tissue, skeletal muscle, myocardium and testis. The protein was compared with other hepatic binding-protein preparations of similar molecular weight.

Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus.

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.