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Increased Steroid Responsiveness during Sodium Butyrate-induced “Differentiation” of HeLa S 3 Cells*
Abstract
Pretreatment of HeLa S3 cells with 5 mM sodium n-butyrate markedly enhances cellular responsiveness to the synthetic glucocorticoid dexamethasone, using increased alkaline phosphatase activity as a marker for steroid action. In contrast, dexamethasone pretreatment does not affect the responses of cells to butyrate. Maximal effects of butyrate on steroid responsiveness occur after 2 days of pretreatment. The increased responsiveness of butyrate-pretreated cells to dexamethasone is partially explained by the collection of most cells at a block point in the hormonally responsive portion of the G1 phase of the cell cycle. Cell cycle population effects on steroid responsiveness are lost only gradually over 40 h after the release from butyrate, as cells leave the hormonally responsive late G1 and S phases. In addition to cell cycle population effects, a second, more rapidly reversible effect of butyrate on steroid responsiveness occurs within the late G1 phase itself at the butyrate block point. This second effect is fully and rapidly lost within 10 h after butyrate's removal, a time before the entry of the released cells into S phase. The reversal of butyrate-induced histone hyperacetylation was examined during this 10-h period. Hyperacetylation is lost in less than 2.5 h after butyrate's removal, suggesting that a rapidly reversible enhancement of glucocorticoid action may occur in the late G1 phase when histones are hyperacetylated. This rapidly reversible process appears to be distinct from the more slowly reversible cell cycle population effects.
... HDACi prevented activation of transiently transfected, episomal, or chromosomal MMTV promoters by glucocorticoids (Mulholland et al., 2003;Kinyamu and Archer, 2004). Although sodium butyrate inhibited glucocorticoid induction of the tyrosine aminotransferase gene in rat HTC cells (Plesko et al., 1983), it enhanced glucocorticoid induction of alkaline phosphatase in HeLa S3 cells (Littlefield and Cidlowski, 1984). Finally, trichostatin A induced estrogen-dependent transcription in MCF-7 cells (Ruh et al., 1999) and in stably transfected HepG2 cells (Mao and Shapiro, 2000). ...
... The strong dose-dependent stimulatory effects of HDACi on GRE5-TATA-CAT and endogenous TAT gene expression differ markedly from previously reported results demonstrating down-regulation of the stimulatory effect of glucocorticoids on the MMTV promoter in various cell types (Mulholland et al., 2003;Kinyamu and Archer, 2004) or on the TAT gene in rat hepatoma cells (Plesko et al., 1983). Our results, in contrast, are compatible with earlier observations that sodium butyrate enhances dexamethasone responsiveness of the alkaline phosphatase gene in HeLa S3 cells (Littlefield and Cidlowski, 1984). Although the long time course of induction of glucocorticoid reporter vectors in Ishikawa cells may suggest indirect effects mediated by the altered expression of a component of the glucocorticoid signaling pathway, our assays for GR mRNA and protein levels are not consistent with an induction in GR expression. ...
... The GR has been reported to have differential transcription activity in G 1 and S phases (permissive) and in G 2 /M phases (nonpermissive) (Hsu and DeFranco, 1995;King and Cidlowski, 1998). Long-term (3 day) effects of sodium butyrate on GR activation of the alkaline phosphatase gene in HeLa S3 cells were attributed to synchronization of the cells in the permissive G 1 phase (Littlefield and Cidlowski, 1984). Note, however, that a recent study reported that treatment with 300 nM trichostatin A for 3 days is accompanied by a decrease in the proportion of cells in both the G 0 /G 1 and S phases and an increase in cells in G 2 /M (Takai et al., 2004). ...
Histone deacetylase inhibitors (HDACi), which have emerged as a new class of anticancer agents, act by modulating expression of genes controlling apoptosis or cell proliferation. Here, we compared the effect of HDACi on transcriptional activation by estrogen or glucocorticoid receptors (ER and GR, respectively), two members of the steroid receptor family with cell growth regulatory properties. Like other transcription factors, steroid receptors modulate histone acetylation on target promoters. Using episomal reporter vectors containing minimal promoters to avoid promoter-specific effects, we observed that long-term (24-h) incubation with HDACi strongly stimulated GR-dependent but markedly repressed ER-dependent signaling in ER+/GR+ human endometrial carcinoma Ishikawa cells. These effects were reproduced on endogenous target genes and required incubation periods with HDACi substantially longer than necessary to increase global histone acetylation. Repression of estrogen signaling was due to direct inhibition of transcription from multiple ERalpha promoters and correlated with decreased histone acetylation of these promoters. In contrast, the strong HDACi stimulation of GR-dependent gene regulation was not accounted for by increased GR expression, but it was mimicked by overexpression of the histone acetyltransferase complex component transcriptional intermediary factor 2. Together, our results demonstrate striking and opposite effects of HDACi on ER and GR signaling that involve regulatory events independent of histone hyperacetylation on receptor target promoters.
... SB treatment also increases CEA secretion in the colonic adenocarcinoma cell line (30), HCG in the nontrophoblastic cancer cell line (25) and alpha-fetoprotein in the human hepatic cell line (20). MCF-7 breast carcinoma cells treated with butyrate secreted milk-related antigen (1) and butyrate-induced differentiation caused an increase in steroid responsiveness of HeLa S3 cells (18). Increased estrogen-receptor sites in KC 1-treated nuclei of cultured human endometrial adenocarcinoma (IK) cells has also been reported (22). ...
Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM).
Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment.
Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.
... 22 Interestingly, in rat glioma C6 cells, butyrate was found to block the glucocorticoid-induced increase in glycerol phosphate dehydrogenase 23 but had no effect on glucocorticoid induction of glutamine synthase activity; 24 in HeLa cells the induction of alkaline phosphatase by dexamethasone was, however, potentiated by butyrate. 25 The apparently contradictory effects of butyrate on steroid-mediated gene expression may be explained by a different role of chromatin structure for the regulation of individual genes. A number of studies point to an essential role of chromatin remodelling in the formation of the transcription initiation complex of glucocorticoid-sensitive genes by demonstrating that modulations of nucleosomal positioning and nucleosomal conformation in the promoter region are involved in the transactivation process. ...
In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.
Recent studies have shown that vitamin B6 modulates transcriptional activation by the human glucocorticoid receptor in HeLa S3 cells. We have now examined the possibility that vitamin B6 might similarly influence transcriptional activation by the glucocorticoid receptor in other cell types, as well as gene expression mediated by other members of the steroid hormone receptor superfamily. We show that elevated vitamin B6 concentrations suppress by 40-65% the level of transcription mediated through the endogenous murine L cell glucocorticoid receptor, as well as the human receptor transfected into E8.2 and T47D cells. In contrast, glucocorticoid receptor-mediated transcription was enhanced 60-110% in mild vitamin deficiency. The level of hormone-independent constitutive gene expression was not affected by these same alterations in vitamin B6 concentration. These studies indicated that the transcriptional modulatory effects of the vitamin were neither restricted to specific cell types nor limited to the human form of the glucocorticoid receptor. We next determined if hormone-induced transcription by several other steroid receptors (androgen, progesterone, and estrogen receptors) was analogously affected by alterations in vitamin B6 concentration. Analysis of gene expression mediated through the mouse mammary tumor virus promoter revealed that transcriptional activation of both the androgen and progesterone receptors was reduced by 35-40% under conditions of elevated vitamin B6 and enhanced by 60-90% in deficiency, again under conditions where constitutive expression was unaffected. Using a different promoter, the estrogen-regulated vitellogenin promoter, we found that transcriptional activation of the estrogen receptor was similarly affected. Estrogen-induced gene expression was reduced by 30% under conditions of elevated intracellular vitamin B6 and enhanced by 85% in vitamin deficiency. Thus, vitamin B6 modulates transcriptional activation by multiple classes of steroid hormone receptors. The similarities in vitamin B6 effects on transcription mediated through different promoters, the mouse mammary tumor virus and vitellogenin promoters, suggest that this vitamin may modulate the expression of a diverse array of hormonally responsive genes. These observations together support the hypothesis that vitamin B6 represents a physiological modulator of steroid hormone action.
n-Butyrate was previously found to increase the epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. We show here that butyrate and dexamethasone synergistically modulate the surface expression of the EGF receptors. The butyrate-induced enhancement of high-affinity EGF binding was only slight in the absence of glucocorticoid, but was strongly and dose-dependently amplified by dexamethasone. Butyrate counteracted the inhibition by insulin of the dexamethasone-induced increase in EGF binding. The results indicate that the glucocorticoid has a permissive effect on a butyrate-sensitive process that determines the surface expression of the high-affinity class of EGF receptors.
We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an approximately 50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25-50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucocorticoids have been shown to increase epidermal growth factor (EGF) receptors in HeLa S3 cells via mechanisms dependent upon glucocorticoid receptors. We have now examined the basal and glucocorticoid-induced levels of epidermal growth factor (EGF) receptors in synchronized HeLa S3 cells and related these findings to glucocorticoid receptor nuclear binding, receptor activation, and several physiochemical properties of the glucocorticoid receptor. Quantitation of EGF receptor binding during the cell cycle indicates that no significant variation in EGF receptor number occurs during the cell cycle. Dexamethasone treatment of nonsynchronized HeLa S3 cells results in an approximately 131% increase in EGF receptor number within 48 h of treatment. Administration of glucocorticoids to cells synchronized at the late G1/S phase boundary of the cell cycle results in an approximately 80% increase in epidermal growth factor receptors 8-9 h after treatment. This hormone-induced response disappears as cells enter the G2/M and early G1 phases of the cell cycle. In contrast, hormone administration to synchronized cells during the G2/M phases is without effect after 8 or 9 h, but a response is evident when these cells reenter the late G1 phase. This inability of glucocorticoids to induce EGF receptor binding has been correlated with nuclear glucocorticoid receptor translocation at 37 degrees C in intact cells and activation of receptors in vitro to DNA binding proteins by warming. This reduction in nuclear receptor binding in intact cells and diminished in vitro activation of receptor are associated with the detection of a tightly binding glucocorticoid receptor form as analyzed by hydroxylapatite chromatography. These analyses suggest that the failure of glucocorticoids to induce EGF receptor binding during the G2/M and early G1 phases may be the result of decreased receptor activation which may result from a post-transcriptional modification of the glucocorticoid receptor.
While studying the effects of chemotherapy on glucocorticoid receptor (GR) binding levels in hematological malignancies, we observed a sizable increase in nuclear GR binding of [3H]dexamethasone in peripheral leukocytes from a chronic basophilic leukemia patient following treatment with hydroxyurea plus prednisone, but not after prednisone alone. This apparent clinical effect of hydroxyurea led to an examination of hydroxyurea effects on GR binding and sensitivity in the glucocorticoid-sensitive human lymphoblast cell line GM4672A. GR binding levels in GM4672A cells were measured following a 3-day exposure to 50 microM hydroxyurea, a concentration chosen to have a minimal but measurable effect on cellular growth rates with little or no effect on cellular viability. Under these conditions, nuclear [3H]dexamethasone receptor binding measured by Scatchard analysis using a whole-cell assay was elevated 2.4-fold over control values (P less than 0.05), while cytosolic residual receptor binding (measured at 37 degrees C) remained unchanged. Thus, the total cellular content of measurable GR was increased, and this increase was totally accounted for by GR capable of nuclear binding. Hydroxyurea treatment of GM4672A cells had no effect on the affinity of nuclear or cytosolic GR for [3H]dexamethasone. The increase in measurable nuclear-bound receptors occurred in a time-dependent manner over a period of 3 days and was fully reversible within 3 days following removal of hydroxyurea. The increase in receptor binding could not be explained by the slight alterations in cell cycle kinetics which occur at this low level of hydroxyurea. Despite increased receptor binding, cellular glucocorticoid responsiveness was unaltered as assessed by dexamethasone inhibition of cell growth and dexamethasone inhibition of a urokinase-like plasminogen activator. Thus, increased nuclear and total cellular GR binding levels in hydroxyurea-treated GM4672A cells are not associated with increased glucocorticoid responsiveness.
A previous study described the development of cultures of Nb2 node rat lymphoma cells which specifically require a lactogenic hormone, e.g., prolactin (PRL), as a growth factor. In the present study, sublines of these cultures have been established, including clones, which do not depend on exogenous lactogens for growth; the cells do not appear to produce autocrine, PRL-like substances. Although these autonomous cells grew readily in the complete absence of lactogens, their growth rate was stimulated (up to approximately 30%) by PRL concentrations greater than or equal to 0.1 ng/ml. When cultures of the lactogen-dependent and of the cloned, autonomous lymphoma cells were incubated for 3 days with sodium n-butyrate (NaBT; 2 mM), cells were arrested in the G1 phase of the cell cycle, as shown by flow cytometric analysis. A substantial proportion of the G1-arrested cells was viable and readily resumed growth, within 1 day, when transferred to NaBT-free medium containing PRL (1-100 ng/ml). The resumption of growth of the cells from the lactogen-dependent cultures was critically dependent on PRL; in its absence cells lysed. Surprisingly, the rapid recovery of the cells from the lactogen-independent cultures also depended on the presence of PRL in the medium; in its absence growth did not resume for at least 2.3 days. The acquired need of the NaBT-treated, autonomous cells for PRL was only transient, since such cells reverted fully to the PRL-independent state within about 3 days of culturing in PRL-containing, NaBT-free medium. It is proposed, as a working hypothesis, that the autonomous lymphoma cells can be mitogenically activated by two different pathways, one requiring exogenous lactogens and another which is independent of lactogens; the latter pathway recovers much more slowly from the treatment with NaBT than the lactogen-dependent pathway. This model could explain the sensitivity of the autonomous lymphoma cells to PRL and their transient dependency on exogenous lactogens during their recovery from NaBT treatment. NaBT would appear to be a useful agent for studying the mechanism(s) by which the autonomous lymphoma cells circumvent the need for mitogenic lactogens.
1,25 Dihydroxyvitamin D3 suppressed colony formation in soft agar and increased alkaline phosphatase activity in clonal rat osteosarcoma cells. Sodium butyrate enhanced these effects of the hormone partly through a mechanism involving an alteration of nuclear binding of the hormone. It is suggested that 1,25 dihydroxyvitamin D3 in conjunction with sodium butyrate might be able to regulate differentiation and proliferation of neoplastic cells.
Gene transcription mediated by steroid hormones has become one of the most extensively characterized model systems for studying the regulation of gene expression in eukaryotic cells. However, specific details of gene regulation by steroid hormones are often complex and may be unique in specific cell types. Diverse regulatory mechanisms leading to either activation or repression of particular genes frequently involve interactions between steroid hormone receptors and other ubiquitous and/or cell-specific transcription factors that act on the complex promoter of the regulated gene. Interplay between steroid receptor-mediated and other signal transduction pathways may also be involved. In addition, recent novel results indicate that moderate variations in the intracellular concentration of pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, can have pronounced modulatory effects on steroid-induced gene expression. Specifically, elevation of intracellular PLP levels leads to decreased transcriptional responses to glucocorticoid, progesterone, androgen, or estrogen hormones. Conversely, cells in a vitamin B6-deficient state exhibit enhanced responsiveness to steroid hormones. One aspect of the mechanism by which these transcriptional modulatory effects of PLP occur has recently been shown to involve interruption of functional interactions between steroid hormone receptors and the nuclear transcription factor NF1. These findings--that the vitamin B6 nutritional status of cells modulates their capacity to respond to steroid hormones--impose an additional level of cell-specific control over steroid hormone regulation of gene expression and will serve as the focal point for this review.
The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones.
Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.
Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-kappaB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRalpha), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRbeta), which does not bind known ligands and attenuates GRalpha action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-kappaB DNA binding site 5' to the hGR promoter that leads to a 1.5-fold increase in GRalpha mRNA and a 2.0-fold increase in GRbeta mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-alpha treatment disproportionately increased the steady-state levels of the GRbeta protein isoform over GRalpha, making GRbeta the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-alpha or IL-1. The increase in GRbeta protein expression correlated with the development of glucocorticoid resistance.
To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.
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