Characterization of selective glucocorticoid-dependent responses in a glucocorticoid-resistant smooth muscle tumor cell line

The DDT₁ MF2 smooth muscle cell line was derived from an estrogen/androgeninduced leiomyosarcoma arising in the hamster ductus deferens. Growth of this cell line is arrested in Go/G1 by treatment with glucocorticoids. To facilitate the study of the mechanism of glucocorticoid-induced cell growth arrest, a glucocorticoid-resistant variant cell line, DDT₁ MF2 GR1 (GR1), was developed by genetic selection. Growth of this mutant cell line is completely resistant to the inhibitory action of glucocorticoids. However, we now demonstrate that both primary and secondary glucocorticoid-induced events still exist in the GR1 cell line. By analyzing the expression and genetic pattern of glucocorticoid receptor, no detectable rearrangement of the glucocorticoid receptor gene was found although the expression of both mRNA and protein levels of the receptor were lower in the variant compared to wild-type cells. In addition, we found that the expression of two growth-associated genes, Ha-ras and transforming growth factor β1 (TGF-β1) are down-regulated by glucocorticoids in wild-type DDT₁ MF2 cells but not in GR1 cells. These results indicated that the function or activity of glucocorticoid receptor in the GR1 cells is not qualitatively altered. Our data suggest that a lower glucocorticoid receptor level is not the real cause or at least not the single cause for the GR1 cell's loss of sensitivity to the inhibitory action of glucocorticoid. Instead, we postulate the existence of a defect downstream of the primary site of action of glucocorticoid receptor complexes in GR1 cells. © 1993 Wiley-Liss, Inc.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.     

Glucocorticoid-dependent maturation of mouse mammary tumor virus glycoproteins in mouse lymphoma cells: isolation of variants with constitutive viral protein maturation and normal glucocorticoid receptor function

The posttranslational maturation and cell surface localization of mouse mammary tumor virus (MMTV) envelope glycoproteins is regulated by glucocorticoid hormone in mouse T-lymphoma cell line W7MG1. Only when the cells are cultured with glucocorticoid is the MMTV envelope precursor, Pr74, converted efficiently to the two mature proteolytic products, gp52 and gp33. By immunological selection we have isolated protein-processing variants that express the mature viral proteins constitutively on the cell surface. The rate of synthesis of Pr74 is indistinguishable in variant and wild-type cells, but the variants efficiently convert Pr74 to gp52 and gp33 even when grown without the hormone. The variant phenotype persists when the variant cells are fused with uninfected wild-type cells to form somatic cell hybrids, indicating that the variant phenotype resulted from expression of a new or altered function that is not expressed in wild-type cells grown without glucocorticoid. Although the specific gene whose structure or regulation is altered in the variant has not yet been determined, some possibilities have been eliminated. First, the number and function of the glucocorticoid receptors in the variant cells was normal, suggesting that alterations in this protein were not responsible for the variant phenotype. Second, comparison by two-dimensional gel electrophoresis of gp52 produced in variant and wild-type cells revealed no differences in size or charge, indicating no gross differences in the processing of the viral proteins in the variant and wild-type cells.     

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.     

Nuclear glucocorticoid receptor binding in L6 skeletal muscle cells in culture

Mechanisms underlying glucocorticoid hormone actions on skeletal muscle remain incompletely understood. This problem may be amenable to solution with a simple cell culture system in which the hormonal environment can be controlled. In this report, we demonstrate that the L6 muscle cell line may provide such a system. These cells, which possess many morphological and functional characteristics of skeletal muscle, originate as mononuclear myoblasts, which fuse to form multinucleated myotubes. L6 myoblasts and myotubes contain an intracellular glucocorticoid receptor that has binding parameters and ligand specificity similar to those of glucocorticoid receptors of classical glucocorticoid target tissues. A major advantage of the use of cultured cells is ease of isolation of myonuclei that display specific glucocorticoid receptor binding. L6 muscle cells should provide a valuable model system for further studies of the mechanisms of glucocorticoid hormone actions on muscle.     

Dexamethasone Mediated Stabilization of Insulin Receptor mRNA

Abstract

To determine the mechanism of glucocorticoid mediated enhancement of insulin receptor (IR) gene expression, we cotransfected a glucocorticoid receptor expression vector and a plasmid containing a reporter gene driven by an MMTV or IR promoter into COS 7 cells. Dexamethasone (Dex) increased MMTV promoter activity by 100% but had no effect on IR promoter activity. In the glucocorticoid responsive breast cancer cell line, MCF-7, Dex increased IR mRNA by 60%, and increased the IR mRNA half-life from approximately 6hrs to >24hrs. No glucocorticoid responsive element could be located in the insulin receptor 3′ untranslated region. Glucocorticoids stabilize IR mRNA.     

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.     

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.     

A new determinant of glucocorticoid sensitivity in lymphoid cell lines

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoid receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well- characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone due to the absence of another function designated "I" for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild- type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation.     

Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.     

Down-regulation of glucocorticoid receptors in mouse lymphoma cell variants.

The mouse thymoma-derived cell line W7 is sensitive to the cytolytic action of glucocorticoids. We have isolated a novel class of cell variant that apparently overcomes its inherent sensitivity to glucocorticoids by reversibly down-regulating the level of glucocorticoid receptors. This phenotype is stable during subcloning in the presence and in the absence of glucocorticoids and is dominant in somatic cell hybrids with wild-type cells. Fusion of this variant with wild-type cells produces hybrids that down-regulate and are less sensitive to glucocorticoids than hybrids of receptor-negative and wild-type cells. This is the first demonstration of a phenotypic change which correlates with down-regulation of the glucocorticoid receptor.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Functional interaction between the glucocorticoid receptor and GANP/MCM3AP

Glucocorticoids are widely used to treat inflammatory diseases but have a number of side effects that partly are connected to inhibition of cell proliferation. Glucocorticoids mediated their action by binding to the glucocorticoid receptor. In the present study, we have identified by two-hybrid screens the germinal center-associated protein (GANP) and MCM3-associated protein (MCM3AP), a splicing variant of GANP, as glucocorticoid receptor interacting proteins. GANP and MCM3AP can bind to the MCM3 protein involved in initiation of DNA replication. Glutathione-S-transferase-pull-down and co-immunoprecipitation assays showed that the C-terminal domain of GANP, encompassing MCM3AP, interacts with the ligand-binding domain of the glucocorticoid receptor. Characterization of the intracellular localization of GANP revealed that GANP is shuttling between the nucleus and the cytoplasm. Furthermore, we show that glucocorticoids are unable to inhibit DNA replication in HeLa cells overexpressing MCM3AP suggesting a role for both glucocorticoid receptor and GANP/MCM3AP in regulating cell proliferation.