Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BACKGROUND: Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. METHODS: We analyzed the effect of treatment with the cytosine analogue 5 aza-2' deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. CONCLUSIONS: Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

Role of c-Myb in the survival of pre B-cell acute lymphoblastic leukemia and leukemogenesis.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The current treatment protocol for ALL involves an intense chemotherapy regimen yielding cure rates of nearly 80%. However, new therapies need to be designed not only to increase the survival rate but also to combat the risk of severe therapy associated toxicities including secondary malignancies, growth problems, organ damage, and infertility. The c-Myb proto-oncogene is highly expressed in immature hematopoietic cells. In this study, we demonstrate that loss of c-Myb itself decreased the viability of these leukemic cells. Additionally, the inhibition of c-Myb caused a decrease in cell proliferation, significantly increased the number of cells in G(0) /G(1) phase of the cell cycle, increased the sensitivity of pre-B-ALL cells to cytotoxic agents in vitro, and significantly delayed disease onset in a mouse model of leukemia. Furthermore, we demonstrate that Bcl-2 is a target of c-Myb in pre-B-ALL cells. Our results identify c-Myb as a potential therapeutic target in pre-B-ALL and suggest that suppression of c-Myb levels or activity, in combination with currently used therapies and/or dose reduction, may lead to a decrease in toxicity and an increase in patient survival rates. Because c-Myb is aberrantly expressed in several other malignancies, targeting c-Myb will have broad clinical applications.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia.

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

c-Myb interacts with the glucocorticoid receptor and regulates its level in pre-B-acute lymphoblastic leukemia cells.

Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells.

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells.

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.

Use of recombinant cell-permeable small peptides to modulate glucocorticoid sensitivity of acute lymphoblastic leukemia cells.

Glucocorticoid (GC) hormones induce apoptosis in T-cell and pre-B-cell acute lymphoblastic leukemia (ALL) cells. Steroid-mediated apoptosis requires a threshold level of the glucocorticoid receptor (GR) protein, and increasing the intracellular GR levels in ALL cells would augment their hormone sensitivity. A protein transduction domain (PTD) approach was used to accomplish this. We produced an HIV Tat PTD domain fusion protein (Tat-GR(554-777)) that potentially competes for the degradation of GR protein by the ubiquitin-proteasome system and should thus increase its intracellular levels by "stabilizing" the GR. We also designed a fusion peptide for the c-Myb DNA binding domain, Tat-c-Myb DBD, since the biological function of this peptide as a dominant negative inhibitor of the c-Myb protein was already known. Purified, bacterially expressed Tat-c-Myb DBD and Tat-GR(554-777) exhibited highly efficient transduction into cultured ALL cell lines including 697 (pre-B-ALL) and CEM-C7 (T-ALL) cells. As expected, the transduced Tat-c-Myb DBD peptide inhibited steroid-mediated stimulation of a GR promoter-luciferase reporter gene. Significantly, transduced Tat-GR(554-777) effectively increased intracellular GR levels in the GC-resistant T-ALL cell line, CEM-C1, and in the pre-B-ALL 697 cell line. Furthermore, transduction of Tat-GR(554-777) rendered GC-resistant CEM-C1 cells sensitive to steroid killing and further sensitized 697 cells to steroid. The use of Tat-fusion peptide transduction may eventually lead to innovative therapeutic modalities to improve the clinical response of patients suffering from T-cell and pre-B-cell acute lymphoblastic leukemia by increasing steroid responsiveness and perhaps converting steroid-resistant leukemia to a hormone-responsive phenotype.

Glucocorticoid receptor knock down reveals a similar apoptotic threshold but differing gene regulation patterns in T-cell and pre-B-cell acute lymphoblastic leukemia.

Glucocorticoids (GCs) are used in combination therapy for treating acute lymphoblastic leukemia (ALL). In T-cell (CEM-C7) and pre-B-cell (697) ALL cell lines, dexamethasone (Dex) treatment causes an auto-upregulation of glucocorticoid receptor (GR) mRNA transcripts and protein. We hypothesized that there is a threshold level of GR transcripts/protein needed for cells to respond to the apoptosis-inducing effects of hormone. GR knock down using a doxycycline-controllable shRNAmir indicated that the apoptotic response changes from sensitive to resistant with changing GR levels. Titration of the 697 cell GR to equal that of the CEM-C7 T-cell ALL line caused a shift in sensitivity to that seen in CEM-C7 cells. While the same level of GR is required to trigger apoptosis in both T-cell and pre-B-cell ALL lineages, similarities and differences were observed for the regulation of target genes in these lineages. These preliminary gene regulation patterns may lead to the development of a molecular signature for GC-sensitive and GC-resistant leukemia cells.

A conserved molecular mechanism is responsible for the auto-up-regulation of glucocorticoid receptor gene promoters.

Glucocorticoid (GC) hormones are widely used in the treatment of acute lymphoblastic leukemia (ALL). Whereas a high level of GC receptor (GR) protein is associated with the sensitivity of ALL cells to steroid-mediated apoptosis, the auto-up-regulation of human (h)GR mRNA and protein is also found in hormone-sensitive ALL cell lines. We have characterized the hGR gene-proximal promoters for DNA sequences and transcription factors required for hormone responsiveness in T lymphoblasts. Sequences at -4559/-4525 and -2956/-2916, relative to the translation start site, function as strong composite GC response units (GRUs). Both GRUs include adjacent protein recognition sequences for the c-Myb transcription factor and the GR as a DNA cassette. An Ets-binding sequence overlaps the GR-binding site in the -4559/-4525 GRU, whereas an Ets-binding site present in the -2956/-2916 GRU does not overlap the GR/c-Myb-binding cassette. The Ets protein family member, PU.1, blocks hormonal activation of the -4559/-4525 GR/c-Myb-binding cassette but does not interfere with the responsiveness of the -2956/-2916 GRU. Thus, the hGR 1A GRU (described previously), the -4559/-4525 GRU, and the -2956/-2916 GRU have a similar structure and can mediate cell type-specific hormonal auto-up-regulation of hGR promoter activity in steroid-sensitive ALL cells. However, subtle differences in the GRU architecture result in differential sensitivity of the promoters to Ets family members such as PU.1. The architecture of the GRU and the spectrum of specific transcription factors present in different types of ALL might allow the development of a tailored therapy to enhance steroid sensitivity in ALL patients.

c-Myb and members of the c-Ets family of transcription factors act as molecular switches to mediate opposite steroid regulation of the human glucocorticoid receptor 1A promoter.

Steroid auto-regulation of the human glucocorticoid receptor (hGR) 1A promoter in lymphoblast cells resides largely in two DNA elements (footprints 11 and 12). We show here that c-Myb and c-Ets family members (Ets-1/2, PU.1, and Spi-B) control hGR 1A promoter regulation in T- and B-lymphoblast cells. Two T-lymphoblast lines, CEM-C7 and Jurkat, contain high levels of c-Myb and low levels of PU.1, whereas the opposite is true in IM-9 B-lymphoblasts. In Jurkat cells, overexpression of c-Ets-1, c-Ets-2, or PU.1 effectively represses dexamethasone-mediated up-regulation of an hGR 1A promoter-luciferase reporter gene, as do dominant negative c-Myb (c-Myb DNA-binding domain) or Ets proteins (Ets-2 DNA-binding domain). Overexpression of c-Myb in IM-9 cells confers hormone-dependent up-regulation to the hGR 1A promoter reporter gene. Chromatin immunoprecipitation assays show that hormone treatment causes the recruitment of hGR and c-Myb to the hGR 1A promoter in CEM-C7 cells, whereas hGR and PU.1 are recruited to this promoter in IM-9 cells. These observations suggest that the specific transcription factor that binds to footprint 12, when hGR binds to the adjacent footprint 11, determines the direction of hGR 1A promoter auto-regulation. This leads to a "molecular switch" model for auto-regulation of the hGR 1A promoter.

Human glucocorticoid receptor alpha transcript splice variants with exon 2 deletions: evidence for tissue- and cell type-specific functions.

Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. When fused to Flag and enhanced green fluorescent protein (EGFP) tags at the carboxy terminus, all transcript variants can be correctly translated in vitro and in vivo. The Flag-tagged hGRalpha protein variants can functionally bind to a glucocorticoid response element (GRE) and can mediate hormonal stimulation of a pGRE-luciferase reporter gene. Compared to the "classical", native hGRalpha, these four variants exhibit a cell type-specific activation of a reporter gene, and this is influenced by the hGRalpha 3' untranslated region in the hGR transcript. When equal amounts of the cDNAs for these GRalpha variant proteins are transfected into cells, they can exhibit lower or higher transcriptional activation compared to the classical GR. Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.

Interaction between the interferon signaling pathway and the human glucocorticoid receptor gene 1A promoter.

The newly described 1A promoter of the human glucocorticoid receptor (hGR) gene contains an interferon (IFN) regulatory factor element (IRF-E), a binding motif for the family of proteins termed IFN regulatory factors (IRFs) that are regulated by IFNs. To examine the in vivo role of IFNs in hGR gene regulation, human T cell lines (CEM-C7 and Jurkat) were treated with IFN gamma. IFN gamma rapidly induces the expression of IRF-1 proteins in a dose- and time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A promoter IRF-E after IFN gamma treatment, whereas IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1 and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however, only IRF-1 activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in T cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Additional studies revealed that the glucocorticoid steroid hormone, dexamethasone (DEX), completely blocks IFN induction of IRF-1 mRNA levels. This could explain the lack of any greater apoptotic response to a combination of DEX plus IFN compared with the response to DEX alone. In addition, treatment with IFN gamma alone does not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter) in T lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1-driven transcription between the hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such as methylation.

Three mechanisms are involved in glucocorticoid receptor autoregulation in a human T-lymphoblast cell line.

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.

Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter.

At least three promoters (1A, 1B, and 1C) control the expression of mRNA transcripts for the human glucocorticoid receptor (hGR) protein. An hGR 1A promoter/exon sequence (-218/+269) contains at least 12 deoxyribonuclease (DNase) I footprints that contain bound protein. Whereas four of these footprints (FP6, FP7, FP8, and FP11) contain bound hGR in protein-DNA complexes that are formed, only two (FP7 and FP11) appear to be important for the up-regulation of hGR 1A promoter/exon activity in T-lymphoblasts. Furthermore, the activity of these DNA elements depends upon the promoter context, leading to a redundant and complex regulation of expression of the hGR 1A promoter/exon. FP7 appears to be required for hormonal responsiveness in the absence of upstream sequences (+41/+191), whereas the hormonal responsiveness of FP11 requires a functional, adjacent FP12 DNA sequence. FP12 contains overlapping binding sites for the protooncogene transcription factors c-Myb and c-Ets. It seems likely that binding of either c-Myb or c-Ets to FP12 is necessary for the direct or indirect binding of the hGR to FP11 (a nonconsensus glucocorticoid response element), and the resultant steroid-responsiveness of the hGR 1A promoter/exon sequence. We propose that the identity of the accessory transcription factor bound to FP12 (c-Myb or c-Ets) may determine the nature of regulation (positive or negative) of hGR gene expression by hormone, and that this may be important for hormone-induced apoptosis in T cell acute lymphoblastic leukemia.

Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines.

We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.

Characterization of promoter 1B in the human glucocorticoid receptor gene.

At least three different promoter regions (1A, 1B, and 1C) are involved in the expression of the human GR gene. Promoters 1B and 1C are found in a 2800 bp region of DNA immediately upstream of the exon 1C transcriptional initiation site. Transcripts containing either exon 1B or 1C are expressed in a wide variety of human tissues and cultured cells. Luciferase reporter constructs were created containing promoter 1B plus 1C (-2738 to +19), promoter 1B (-2738 to -1046) alone, or promoter 1C (-1045 to +19) alone. All three constructs were equally effective in driving luciferase expression in HeLa (human cervical carcinoma) cells. In Jurkat (human T-cell acute lymphoblastic leukemia) cells, constructs containing promoters 1B plus 1C or promoter 1B were equally active, but the promoters 1B plus 1C construct was 35% more active than the promoter 1C construct. However, in HepG2 (human hepatoma) cells, the promoter 1C construct was as effective as promoters 1B plus 1C and more than twice as effective as promoter 1B. Sequences that reside proximal to the exon 1B transcriptional start site included three Sp1 (FP2-FP4) sites. Another site (FP1) contains the sequence TGATAG, which strongly resembles the consensus binding sequence for the GATA family of transcription factors. However, oligonucleotide competition and supershift analysis of FP1 indicates that this site is not a binding site for GATA proteins. These four sites are in addition to three YY1 and one Sp1 sites previously reported in promoter 1B. In HeLa cells, deletion of the three YY1 sites results in only a 30% loss of activity and substantial loss of activity occurs only after deletion of all four Sp1 sites, indicating the critical importance of Sp1 in GR expression in these cells. In contrast, the elimination of the three YY1 sites results in a dramatic decrease in promoter strength in both HepG2 and Jurkat cells (64 and 77%, respectively), while subsequent deletions of promoter elements do not result in substantial changes in promoter activity in these cell lines. This study shows that both promoters 1B and 1C are important for the ubiquitous expression of the human GR gene. Differences in the utilization of these promoters in various cell types are likely a reflection of different promoter availability and/or the levels of specific transcription factors in the cell. This could contribute to tissue-specific expression of GR levels in different cell types.