Biomedical subjects

R L Miesfeld

Publications and source records attributed to R L Miesfeld.

At least 19 recordsLinked to original sources

PubMed2004

Differential control of eosinophil survival by glucocorticoids.

Glucocorticoids are effective drugs for eosinophil-related disorders, such as asthma and allergy. Previous studies have demonstrated that glucocorticoids increase eosinophil apoptosis and block the survival effect of submaximal concentrations of interleukin-5 (IL-5). We investigated the effect of glucocorticoids on eosinophil survival in the presence of a higher concentration of IL-5 (1 ng/ml), comparable to IL-5 levels in bronchoalveolar lavage and sputum specimens from patients with asthma. In contrast to incubation in the presence of submaximal concentrations of IL-5, the addition of dexamethasone (DEX) to media containing 1 ng/ml IL-5 led to a significant increase in eosinophil cell viability from 58 +/- 6.9% to 87 +/- 2.4% ( p < 0.005) after 72 hours in culture. We found that RU486 blocked the DEX effect on cell viability confirming that glucocorticoid receptor functions are required. We investigated the possibility that the glucocorticoid enhancement of eosinophil survival may be due to an effect on IL-5 receptor expression. Our results show that the IL-5 associated decrease in IL-5 receptor alpha-subunit expression was blocked significantly after 24 hrs in culture with media containing IL-5 plus DEX compared to IL-5 alone. It is tempting to speculate that the observed glucocorticoid enhancement of eosinophil survival in the presence of elevated concentrations of IL-5 could be a mechanism that contributes to glucocorticoid resistance in asthma.

Adult↗

PubMed2001

Analysis of glucocorticoid and androgen receptor gene fusions delineates domains required for transcriptional specificity.

Androgen receptor (AR) and glucocorticoid receptor (GR) influence distinct physiologic responses in steroid-responsive cells despite their shared ability to selectively bind in vitro to the same canonical DNA sequence (TGTTCT). While the DNA-binding domains (DBDs) of these receptors are highly conserved, the amino N-terminal domain (NTD) and hormone-binding domain (HBD) are evolutionarily divergent. To determine the relative contribution of these functional domains to steroid-specific effects in vivo, we constructed a panel of AR/GR gene fusions by interchanging the NTD, DBD, and HBD regions of each receptor and measured transcriptional regulatory activities in transfected kidney and prostate cell lines. We found that GR was approximately 10-fold more active than AR when tested with the mouse mammary tumor virus promoter, and that this difference in activity was primarily owing to sequence divergence in the NTDs. We also tested transcriptional activation of the androgen-dependent rat probasin promoter, and in this case, AR was at least twofold more active than GR. Analysis of the chimeric receptors revealed that this difference mapped to the DBD region of the two receptors. Transcriptional repression functions of the wild-type and chimeric receptors were measured using an activator protein 1 (AP-1) transrepression assay and identified the GR HBD as a more potent transrepressor of AP-1 transcriptional activation than the AR HBD. Taken together, our analyses reveal that evolutionary sequence divergence between AR and GR functional domains results in unique promoter-specific activities within biologic systems in which both AR and GR are normally expressed.

Amino Acid Sequence↗

PubMed1999

Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis.

Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.

Animals↗

PubMed1998

Glucocorticoid regulation of GM-CSF: evidence for transcriptional mechanisms in airway epithelial cells.

Inflammation plays a central role in the pathogenesis of asthma. Glucocorticoids are first-line anti-inflammatory therapy in the treatment of asthma and are effective inhibitors of inflammatory cytokines. Clinical data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) production by airway epithelial cells may be an important target of inhaled glucocorticoid therapy. We examined the regulatory mechanisms of GM-CSF expression by interleukin-1beta (IL-1beta) and the synthetic glucocorticoid dexamethasone in the BEAS-2B human bronchial epithelial cell line. IL-1beta stimulation resulted in a 15-fold induction of GM-CSF protein, which was associated with a corresponding 47-fold maximal induction of GM-CSF mRNA levels. Treatment with the transcriptional inhibitor actinomycin D before IL-1beta stimulation completely abolished induction of GM-CSF mRNA, whereas incubation with cycloheximide had no effect. Taken together, these data demonstrate that IL-1beta induction of GM-CSF is mediated through transcriptional mechanisms. Dexamethasone treatment of BEAS-2B cells produced an 80% inhibition of IL-1beta-induced GM-CSF protein and a 51% inhibition of GM-CSF mRNA. GM-CSF mRNA was rapidly degraded in these cells, and dexamethasone treatment did not significantly affect this decay rate. We conclude that, in the BEAS-2B bronchial epithelial cell line, IL-1beta induction and dexamethasone repression of GM-CSF expression are mediated predominantly through transcriptional mechanisms.

Bronchi↗

PubMed1997

Crosstalk during Ca2+-, cAMP-, and glucocorticoid-induced gene expression in lymphocytes.

In the WEHI7.2 thymoma cell line, cAMP, glucocorticoids, or increases in cytosolic Ca2+ concentration lead to cell death by apoptosis. In the present study, we examined the effects of these compounds on cAMP response element (CRE)-mediated gene expression. Thapsigargin and A23187 were employed to increase cytosolic Ca2+ levels and induce apoptosis. Both compounds enhanced transcription from a CRE preceding apoptotic death. Moreover, the transcriptional response to the combination of forskolin and either thapsigargin or A23187 was synergistic mirroring the effect on cell death. Importantly, dexamethasone treatment, which causes an efflux of Ca2+ from the ER, induced transcription from a CRE alone or in synergy with forskolin. The increase in CRE-controlled gene expression correlated with a decrease in cell viability. Following treatment with forskolin, thapsigargin, or dexamethasone, the CRE binding protein (CREB) was phosphorylated at levels correlating with the level of induced gene expression. These data suggest that transcriptional crosstalk between independent signaling pathways occurs in lymphocytes, and CREB may play a central role in the mediation of CRE-dependent transcription by these diverse set of apoptotic agents.

Animals↗

PubMed1997

Glucocorticoid receptor signaling in a bronchial epithelial cell line.

Glucocorticoids are an effective anti-inflammatory therapy for the treatment of asthma. The anti-inflammatory effects of glucocorticoids may be due to the inhibition of transcription factors that regulate cytokine synthesis. Because of the potential role of the bronchial epithelium in asthmatic inflammation and the possibility that this cell may be the main target of inhaled glucocorticoids, we have characterized glucocorticoid receptors (GR) and GR signaling in the human bronchial epithelial cell line BEAS-2B. Western blot analysis and radioligand binding studies demonstrated that BEAS-2B cells have functional GR that bind to dexamethasone (Dex) (dissociation constant = 5.6 nM and maximal density of binding sites = 228 +/- 3.3 fmol/mg protein). GR were activated by Dex as assessed using a glucocorticoid-responsive reporter plasmid. Transfection of BEAS-2B cells with an activator protein-1 (AP-1) reporter construct followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment resulted in a fivefold induction of reporter gene activity. Transfection with a nuclear factor (NF)-kappa B reporter construct followed by tumor necrosis factor-alpha (TNF-alpha) treatment resulted in a 10-fold induction of reporter gene activity. Dex (10(-7) M) markedly repressed both the induced AP-1 and NF-kappa B activity. The GR antagonist RU-486 inhibited the repressive effect of Dex on TNF-alpha-induced NF-kappa B activity by 81% but only counteracted the repressive effect of Dex on TPA-induced AP-1 activity by 43%. These studies demonstrate that cross-signaling between AP-1 and NF-kappa B with GR may explain the anti-inflammatory properties of glucocorticoids in airway epithelial cells.

Bronchi↗

PubMed1996

Delineation of two distinct type 1 activation functions in the androgen receptor amino-terminal domain.

Based on the finding that some transcription factors contain multiple transcriptional regulatory activities, we constructed a panel of rat androgen receptor (AR) mutants containing small internal deletions and point mutations within the amino-terminal region of the receptor. Trans-activation assays in CV-1 cells using AR-responsive reporter genes were performed and led to the identification of two noncontiguous trans-activation regions in the AR amino terminus. One of these regions, termed activator function 1a (AF-1a) is a highly-conserved 14-amino acid segment that is predicted to form a beta-turn followed by an acidic amphipathic alpha-helix. Point mutagenesis within AF-1a revealed that two adjacent hydrophobic residues were required for full AR trans-activation function, as arginine substitutions resulted in a 60% reduction in transcriptional activity. A second amino-terminal region was also identified and has been designated AF-1b. Deletion of the 65-amino acid AF-1b segment, which contains numerous glutamate and aspartate residues, caused a 55% decrease in trans-activation function. An AF-1a/AF-1b double mutant retains less than 10% trans-activation function compared with wild-type AR, suggesting that AF-1a and AF-1b may each contribute separately to maximal AR activity. To determine whether AF-1a and AF-1b play a role in AR-mediated trans-repression of AP-1 function, we tested single and double AF-1a/AF-1b mutants in a transient trans-repression assay. Our results showed that neither AF-1a nor AF-1b was required for AP-1 trans-repression, demonstrating that AR-mediated trans-repression and trans-activation are discrete functions.

Amino Acid Sequence↗

PubMed1996

Transcriptional control of steroid-regulated apoptosis in murine thymoma cells.

Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor (GR) N-terminal domain are required for glucocorticoid-mediated apoptosis. However, more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism. To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis, we stably transfected GR, GR variants, and the androgen receptor (AR) into receptor-negative S49 murine thymoma cells. GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway, and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus (MMTV) LTR reporter gene was observed. Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction, thus supporting a role for transactivation in apoptosis. In contrast, we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone, despite its relative inability to induce high level expression of MMTV. To investigate this further, we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18. We found that GIG18 was rapidly induced to comparable levels by both AR and GR, demonstrating that AR can indeed function as a transcriptional activator in S49 cells and, moreover, that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells. Taken together, these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis, although an additional role for GR-mediated transrepression cannot be excluded.

Animals↗

PubMed1995

Isolation of differentially expressed sequence tags from steroid-responsive cells using mRNA differential display.

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3' end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.

Androgens↗

PubMed1995

Adenomatous polyposis coli, protein kinases, protein tyrosine phosphatase: the effect of sulindac.

A putative explanation of the effect of sulindac on adenomatous colon and duodenal polyps from clinical observations and related in vitro experiments is presented. In cells with mutant APC genes, persistent high prostaglandin content of polyps leads to desensitization, downregulation of adenylate cyclase, uncoupling of cAMP synthesis from prostaglandin, and inactivation of protein kinase A (PKA). It is suggested that in normal cells, (APC) protein binds to catenins and microtubules to maintain structure and contribute to cell-cell communication, adherence, and the dephosphorylated state, a necessary condition for such functions. Cells with mutant APC product become isolated, deprived of communication and adhesion to other epithelial cells, overphosphorylated, and without corrective capability. The latter is largely due to downregulation of cAMP synthesis and protein kinase A activity secondary to high prostaglandin. Three main biochemical defects ensue: (1) the restrictive influence of PKA catalyzed phosphorylation of Raf-1 kinase and resultant effects on the MAP kinase cascade and transcription is lost, (2) the transcription of immediate early genes, including cyclooxygenase is stimulated, and (3) the stimulation of protein tyrosine phosphatase (PTPase) by PKA is in abeyance. These putative abnormalities are reversed by inhibition of cyclooxygenase-1 by sulindac. cAMP synthesis and PKA activity return to normal. PKA catalyzed phosphorylations block Raf-1 kinase at the confluence of the Ras and protein kinase C pathways. The MAP kinase cascade is inhibited as is transcription of immediate early genes. At the same time PKA stimulates PTPase, which dephosphorylates the cytoskeleton and restores cell-cell communication, adherence, and structure. The transformed phenotype is circumvented by adjustment of the phosphorylation state and mutant cells rejoin the epithelial community. The redox state of cytoplasm in mutant cells may be shifted toward reduction.

Adenomatous Polyposis Coli↗

PubMed1995

A cell-specific and selective effect on transactivation by the androgen receptor.

The androgen (AR) and glucocorticoid receptors (GR) are related ligand-activated transcriptional regulators which bind the same cis-acting element and are coexpressed in a variety of cell types. Despite a shared DNA binding site, these receptors mediate diverse cellular responses. To explain this paradox, the existence of cell-specific factors that interact with, and modulate the function of, distinct receptors has been proposed. Prostate epithelial cell growth is sensitive to androgens, but is not affected by glucocorticoids, even though both AR and GR are expressed in these cells. We have recently isolated a unique panel of prostate epithelial cell lines from normal rats and have used these cell lines to examine cell-specific steroid responses. In this study, we compared the abilities of AR and GR to enhance transcription of several different reporter genes regulated by simple (i.e., noncompsite) hormone response elements (HREs) in prostate and nonprostate cell lines. The cell-specific effect occurred independently of the AR hormone binding domain and could be observed with a GAL4 fusion protein containing only the AR N-terminal regulatory domain. Gel shift analyses showed that the relative DNA binding affinity of AR for a probe containing a simple HRE was similar in prostate and nonprostate cell extracts. Presently, the only factors known to mediate steroid receptor-specific gene regulation are cJun and cFos, but there were no cell-specific differences in the functional levels of these proteins which could account for a preferential effect on AR-dependent transcription. Taken together, these results suggest that cell-specific activities exist which can preferentially modulate transcriptional transactivation by AR.

Animals↗

PubMed1995

Quantitative differences in androgen and glucocorticoid receptor DNA binding properties contribute to receptor-selective transcriptional regulation.

Androgen receptor (AR) and glucocorticoid receptor (GR) belong to the same subfamily of steroid/nuclear receptors and have been shown to bind qualitatively to the same hormone response element (HRE) DNA sequences. Despite this similarity in target gene recognition, AR and GR have differential affects on the transcriptional regulation of genes containing both simple and complex HRE control regions. Using HREs from the mouse mammary tumor virus (MMTV), tyrosine aminotransferase (TAT), prostatein (C3) or sex-limited protein (SLP) genes, linked to the thymidine kinase promoter, we found receptor-selective differences in the ability of rat AR and rat GR to induce transcription of these various reporter genes. Since AR and GR have a 20% amino acid sequence difference in their DNA binding domains (DBDs), which could result in altered DNA binding affinities, we measured the ability of purified AR and GR DBDs to bind selectively and with high affinity to these HRE sequences in vitro. Gel shift mobility assays showed that the GR DBD had a higher affinity for a consensus HRE than did the AR DBD, and quantitative DNase I footprinting revealed that AR and GR DBDs bound to the MMTV, TAT, C3 and SLP HREs with different affinities. It was found that AR had a dissociation constant (Kd) that was 2-3 times higher than GR on the TAT, C3 and SLP HREs and that the Kd of AR for the C3 and SLP HREs differed by an order of magnitude (43 nM and 460 nM, respectively). Taken together, these data suggest that amino acid differences in the AR and GR DBDs contribute to altered receptor-DNA interactions, however it is likely that non-receptor factors are involved in further modulating receptor-selective DNA binding and transactivation functions.

Amino Acid Sequence↗

PubMed1994

The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function.

Some transcription factors contain stretches of polyglutamine encoded by repeats of the trinucleotide CAG. Expansion of the CAG repeat in the androgen receptor (AR) has been correlated with the incidence and severity of X-linked spinal and bulbar muscular atrophy (Kennedy's disease). In order to understand the relationship of this mutation to AR function, we constructed ARs that varied in the position and size of the polyglutamine tract, and assayed for the abilities of these mutant receptors to bind androgen and to activate transcription of several different AR-responsive reporter genes. Elimination of the tract in both human and rat AR resulted in elevated transcriptional activation activity, strongly suggesting that the presence of the polyglutamine tract is inhibitory to transactivation. Progressive expansion of the CAG repeat in human AR caused a linear decrease of transactivation function. Importantly, expansion of the tract did not completely eliminate AR activity. We postulate that this residual AR activity may be sufficient for development of male primary and secondary sex characteristics, but may fall below a threshold level of activity necessary for normal maintenance of motor neuron function. This functional abnormality may be representative of other genetic diseases that are associated with CAG expansion mutations in open reading frames, such as spinocerebellar ataxia type I and Huntington's disease.

Animals↗

PubMed1994

Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes.

BCL-2 is a 26-kDa integral membrane protein that represses apoptosis by an unknown mechanism. Recent findings indicate that Ca2+ release from the endoplasmic reticulum (ER) mediates apoptosis in mouse lymphoma cells. In view of growing evidence that BCL-2 localizes to the ER, as well as mitochondria and the perinuclear membrane, we investigated the possibility that BCL-2 represses apoptosis by regulating Ca2+ fluxes through the ER membrane. A cDNA encoding BCL-2 was introduced into WEHI7.2 cells and two subclones, W.Hb12 and W.Hb13, which express high and low levels of BCL-2 mRNA and protein, respectively, were isolated. WEHI7.2 cells underwent apoptosis in response to treatment with the glucocorticoid hormone dexamethasone, whereas W.Hb12 and W.Hb13 cells were protected from apoptosis, revealing a direct relationship between the level of BCL-2 expression and the degree of protection. Significantly, BCL-2 also blocked induction of apoptosis by thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump. TG completely inhibited ER Ca2+ pumping in both WEHI7.2 and W.Hb12 cells, but the release of Ca2+ into the cytosol after inhibition of ER Ca2+ pumping was significantly less in W.Hb12 cells than in WEHI7.2 cells, indicating that BCL-2 reduces Ca2+ efflux through the ER membrane. By reducing ER Ca2+ efflux, BCL-2 interfered with a signal for "capacitative" entry of extracellular Ca2+, preventing a sustained increase of cytosolic Ca2+ in TG-treated cells. These findings suggest that BCL-2 either directly or indirectly regulates the flux of Ca2+ across the ER membrane, thereby abrogating Ca2+ signaling of apoptosis.

Apoptosis↗

PubMed1994

Recessive mutations in a common pathway block thymocyte apoptosis induced by multiple signals.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls genes necessary to initiate glucocorticoid-induced thymocyte apoptosis. We have performed a genetic analysis of thymocyte cell death by isolating and characterizing a panel of GR+ dexamethasone-resistant mutants of the murine WEHI7.2 thymocyte cell line. These apoptosis-defective (Apt-) mutants were used to identify previously unknown early steps in the apoptotic pathway. The Apt- mutants contain nonglucocorticoid receptor, recessive mutations in genes that represent multiple complementation groups. These mutations block apoptosis induced by dexamethasone, gamma irradiation, and c-AMP treatment before the point where Bcl-2 exerts its protective effect. We propose that different signals share a common apoptotic pathway, and that the induction of apoptosis involves multiple precommitment steps that can be blocked by recessive mutations.

Animals↗

PubMed1993

Elevated glutathione S-transferase gene expression is an early event during steroid-induced lymphocyte apoptosis.

Based on the finding that glutathione S-transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen-ablated rats, we analyzed GST transcript levels during steroid-induced lymphocyte cell death. It was found that GST gene expression was induced in steroid-sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose-dependent with regard to hormone. GST expression was not induced in an apoptosis-defective variant that contained normal levels of functional receptor, indicating that GST up-regulation was the result of secondary events that occur during steroid-mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroid-sensitive and steroid-resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid-mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up-regulation. Taken together, these results suggest that GST induction in dexamethasone-treated T-lymphocytes occurs early in the steroid-regulated apoptotic pathway and that this may be a marker of calcium-stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway.

Animals↗

PubMed1992

Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens.

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.

Acid Phosphatase↗

PubMed1992

Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis.

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

Animals↗