Regulation of mouse mammary tumor viral RNA synthesis in embryonal carcinoma cells and in teratocarcinoma derived myoblasts.

The expression of the mouse mammary tumor virus (MMTV) was studied in undifferentiated embryonal carcinoma cells (ECC), in partially differentiated myoblastic cells derived from ECC cells, and in fully differentiated myotubes. Whereas no appreciable amount of MMTV RNA could be detected in embryonal carcinoma cells, hybridation with radioactive viral cDNA revealed relatively large quantities of tumor virus RNA in the teratocarcinoma derived myoblasts. The MMTV RNA level was strongly reduced after differentiation of myoblasts into myotubes. The glucocorticoid hormone dexamethasone which stimulates the MMTV RNA synthesis in differentiated mammary cells did not affect this synthesis in myoblastic cells. By contrast, the apparently repressed synthesis of MMTV RNA in myotubes was almost completely overcomed with dexamethasone.     

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.     

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

Dual regulation of protein maturation in viral infected rat HTC hepatoma cells by glucocorticoids and progesterone

Dexamethasone, a synthetic glucocorticoid, is required for full posttranslational maturation of mouse mammary tumor virus (MMTV) phosphoproteins and glycoproteins in M1.54 cells, a viral infected rat hepatoma (HTC) cell line. Pulse-chase radiolabeling with [35S]methionine revealed that steroids with known glucocorticoid activity (such as dexamethasone and hydrocortisone) regulated the maturation of both MMTV polyproteins in a manner proportional to their occupancy for glucocorticoid receptors and their biological potency. In contrast, progesterone selectively induced the proteolytic processing of MMTV phosphoproteins but simultaneously antagonized the dexamethasone-regulated maturation of MMTV glycoproteins and all other tested glucocorticoid responses. Exposure to suboptimal concentrations of both progesterone and dexamethasone fully stimulated the processing of MMTV phosphoproteins, suggesting that steroid receptors occupied with combinations of either steroid functionally interact at the putative maturation gene. Moreover, treatment with either actinomycin D, a potent inhibitor of de novo RNA synthesis, or RU38486, a synthetic antagonist of glucocorticoid and progesterone action, prevented both the dexamethasone and progesterone-regulated induction of MMTV phosphoprotein maturation. Sedimentation velocity and saturation binding analysis revealed that the sizes and concentrations of hepatoma cell progesterone and dexamethasone binding activities are similar while specific binding of the active progestin R5020 was not detected in either M1.54 cells or the glucocorticoid receptor deficient HTC cell line MSN6.10.2. Taken together, our results demonstrate that two distinct classes of steroid hormones can uniquely alter the posttranslational maturation of a specific subset of phosphoprotein substrates by a common glucocorticoid receptor-dependent process.     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.     

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.     

ADP-ribosylation of chromosomal proteins and mouse mammary tumor virus gene expression. Glucocorticoids rapidly decrease endogenous ADP-ribosylation of nonhistone high mobility group 14 and 17 proteins

The relationship between endogenous ADP-ribosylation of chromosomal proteins and glucocorticoid-regulated mouse mammary tumor virus gene expression was investigated in cultured mouse mammary tumor cells. It was observed that glucocorticoids quickly decreased endogenous (ADP-ribose)n on the nonhistone high mobility group (HMG) 14 and 17 proteins. The half-time for this loss was 8 and 17 min, respectively, for the two proteins. (ADP-ribose)n on HMG 1 and 2 and on histone H1 was less susceptible to hydrolysis during glucocorticoid treatment. The rapid loss of (ADP-ribose)n from HMG 14 and 17 occurred in the same time frame as the induction of mouse mammary tumor virus RNA synthesis by glucocorticoids in these cells (Young, H. A., Shih, T. Y., Scolnick, E. M., and Parks, W. P. (1977) J. Virol. 21, 139-149). 3-Amino-benzamide, a specific inhibitor of (ADP-ribose)n synthetase, increased mouse mammary tumor virus RNA levels with an accompanying decrease in endogenous ADP-ribosylation of HMG 14 and 17. These results show that a decrease in endogenous ADP-ribosylation of HMG 14 and 17 is a consequence of glucocorticoid action and suggest that loss of (ADP-ribose)n from these proteins may be an important event in mouse mammary tumor virus gene expression.