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NFκB modulators in a model of glucocorticoid resistant, childhood acute lymphoblastic leukemia
Leukemia Research 34 (2010) 1366–1373
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NFB modulators in a model of glucocorticoid resistant, childhood acute lymphoblastic leukemia Lindsay Nicholson, Andrew G. Hall, Christopher P. Redfern, Julie Irving ∗ Northern Institute for Cancer Research, Newcastle University, Paul O’Gorman Building, Framlington Place, Newcastle upon Tyne, Tyne and Wear, NE2 4HH, UK
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Article history: Received 23 October 2009 Received in revised form 19 December 2009 Accepted 19 December 2009 Available online 27 January 2010 Keywords: Glucocorticoid resistance Childhood acute lymphoblastic leukaemia NFB
a b s t r a c t Glucocorticoids (GCs) are pivotal agents in the treatment of childhood acute lymphoblastic leukaemia (ALL) but the molecular basis of GC-resistance remains unclear. Expression-array studies have shown that commonly upregulated genes associated with GC-sensitivity include GR, glucocorticoid-induced leucine zipper (GILZ) and IB␣, which all negatively interact with components of the pro-survival NFB pathway and therefore may be critical determinants of GC-sensitivity. We have investigated these regulators and their effect on NFB activity in GC-resistant descendents of the B-lineage ALL cell line, PreB 697. We show that while differential up regulation of the modulators (GILZ, GR and IB␣) was demonstrated in GCsensitive compared to GC-resistant sub-lines, this was not coupled with altered nuclear translocation or functionality of the RelA, p50 or c-Rel subunits of NFB. Thus, GC-resistance in the PreB 697 cell line model is not mediated by NFB, however further investigation of the impact of these GC-sensitive associated proteins on other survival pathways, such as the RAS-RAF-MEK-ERK pathway, is warranted. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Glucocorticoids are central to the treatment regime of all lymphoid malignancies, including childhood acute lymphoblastic leukaemia (ALL), due to their ability to induce apoptosis in immature lymphoid cells. The effects are mediated by the glucocorticoid receptor (GR, also known as NR3C1), a ligand-activated transcription factor belonging to the nuclear receptor superfamily. In an unstimulated cell, the GR is located within the cytoplasm bound to a heteromeric complex of heat shock proteins and immunophilins, upon ligand binding the GR undergoes nuclear translocation, dimerizes and can influence gene expression in several ways. The GC–GR complex can bind to DNA and trigger transactivation or transrepression of glucocorticoid response elements (GRE) in target genes [1]. Additionally, the GC–GR complex can indirectly influence gene transcription by protein–protein interactions with other transcription factors, for example, nuclear factor-B (NFB) and AP-1, repressing transcription of their target genes [2]. It is this complex interplay that results in changes in gene expression and ultimately leads to caspase activation and apoptosis in GC-sensitive cells [3]. In childhood ALL, glucocorticoid sensitivity in vitro and in vivo is a significant prognostic indicator and in three consecutive BerlinFrankfurt-Münster (BFM) trials patients responding well to a short mono-therapy GC window were shown to have a better outcome than poor responders [4–6]. Furthermore, leukemic cells of patients
∗ Corresponding author. Tel.: +44 191 246 4369; fax: +44 191 246 4301. E-mail address: [email protected] (J. Irving). 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.12.014
with relapsed ALL are up to 300-fold more resistant to GC in vitro than those taken at diagnosis [7,8] and with relapse rates at 20%, GC-resistance remains a continuing problem. However, despite the inclusion of GCs in treatment protocols for over 50 years, the mechanisms behind defining GC-sensitive or resistant childhood ALL remains elusive [9,10]. Primary patient cells are the obvious choice with which to study GC-resistance in childhood ALL, but spontaneous apoptosis of leukemic blasts in prolonged cell culture and limited amounts of material have made cell line models a valuable alternative. Commonly used GC-resistant, cell line derivatives, such as those of CCRF-CEM and Jurkat, demonstrate loss/mutation of the GR as an acquired GC-resistance mechanism but these events are rarely found in primary ALL cells [11,12] and cannot account for most cases of GC-insensitivity. This disparity is probably due to the mismatch repair deficient status of many ALL cell lines, including CCRF-CEM, since deficiency is associated with an increased basal mutation rate and thus a greater likelihood of acquiring GR mutations under GC selection pressure [13]. In contrast, GC-resistant descendants of a mismatch repair proficient cell lines, PreB 697, predominantly retained two wild-type GR alleles. Again, mismatch repair deficiency, a predominate feature of ALL cell lines, is rarely found in primary ALL samples [14]. A number of studies have used gene expression arrays to try to identify key genes that are transcriptionally regulated in response to GC and may be determinants of GC-sensitivity. Upregulated genes, consistently associated with GC-sensitivity in several studies, included the GR itself, glucocorticoid-induced leucine zipper (GILZ, also known as TSC22D3) and IB␣, (also known as NFB1)
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Fig. 1. Response of PreB 697 cell lines and clinical samples to dexamethasone exposure in vitro. Cell viability was assessed using the MTS assay after a 96-h drug exposure. The viable cell number at each drug concentration was calculated relative to vehicle-control treated cells. Cell line data are the mean ± S.E.M. of at least three independent experiments, patient data are mean ± S.E.M. of at least three wells from a single experiment. (A) GC-sensitivity in the parental PreB 697 and two descendents, R3F9 and R3D11. (B) Phenotypic stability of GC-resistance in the PreB 697 cell lines after early (<5 passages) and late (>15 passages) passage number. (C) GC-sensitivity in a panel of ALL patient samples taken at presentation and relapse (R denotes relapse).
[15–17] all of which have been shown to negatively impact with components of the pro-survival NFB family [18–20]. In unstimulated cells, NFB is sequestered within the cytoplasm, bound to its inhibitory proteins, IB, which block its nuclear translocation. Upon stimulation, for example by TNF␣ exposure, a cascade of signalling events occur, eventually culminating in proteosomal degradation of IB allowing NFB to translocate to the nucleus where it can regulate gene transcription. GR can directly antagonise NFB activity by competing for co-activators or promoter binding [2,21,22] and indirectly by upregulating the synthesis of IkB [23,24]. Further evidence implicating NFB activity in modulating GC-sensitivity comes from a study of paired GC-sensitive and GC-resistant multiple myeloma cell lines in which upregulation of Bruton’s Tyrosine Kinase (BTK) and Hsp27 was associated with GC-resistance [15]. Both of these proteins have been implicated in activating NFB [25,26]. In addition, constitutively activated NF-kappaB has been identified in cALL [27]. The strong correlation between response to GC mono-therapy and clinical outcome is intriguing given that subsequent therapy involves exposure to 6 or 7 other drugs with diverse mechanisms of actions. However, the unifying end response of any chemotherapeutic agent is to trigger cell death. This implies that GC-resistance may be a reflection of the intrinsic propensity of the blasts to undergo apoptosis and that resistance to GC is a more generalised mechanism, affecting response to other therapeutic agents i.e. an increased apoptotic threshold, as might be afforded by variations in NFB signalling. Thus, in this study, we have used the PreB 697 cell lines and two GC-resistant descendents, to investigate GC-related modulators of NFB, their impact on its activity and their relevance to GC-induced apoptosis.
Paisley, UK) supplemented with 10% foetal bovine serum (Invitrogen) at 37 ◦ C and 5% carbon dioxide. The PreB 697, R3F9 and R3D11 cell lines were kindly donated by R. Kofler [13]. The multiple myeloma cell line, RPMI 8226, was purchased from ECACC and cultured in RPMI 1640 supplemented with 10% foetal bovine serum. All cell lines were routinely tested for mycoplasma contamination using MycoAlert® (Lonza, Basel, Switzerland). 2.2. Patient samples Mononuclear cells from bone marrow from untreated children presenting with ALL at the Royal Victoria Infirmary, Newcastle upon Tyne, UK, were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). The cohort consisted of 14 patients (age range, 1.2–18 years), 7 males, 7 females and were predominantly B-lineage (n = 13). Regional ethical committee approval was obtained for the study (reference numbers 2002/111 and 07/H0906). 2.3. Mutational screening GR exon 2 and flanking intronic sequences were amplified from 100–200 ng genomic DNA from all cell lines and were mutationally screened by denaturing HPLC and direct sequencing. Forward and reverse primer sequences, annealing temperatures and denaturing HPLC parameters were performed as previously described [11]. 2.4. In vitro drug sensitivity assays Cell lines or patient cells were plated out in triplicate at 2 × 104 or 2 × 105 , respectively, per well into 96-well plates. Cells were treated with either dexamethasone (Sigma–Aldrich, Dorset, UK) to a range of final concentrations (10−9 to 10−5 M) or control vehicle. Following a 96-h drug exposure, cytotoxicity was assessed using the CellTiter 96 Aqueous One kit (Promega, Southampton, UK), which assesses the capacity of cells to reduce formazan and thus is a measure of metabolically active cells. The resulting absorbances were averaged and expressed as a percentage of the control vehicle. Survival curves were plotted using GraphPad Prism software and IC50 (inhibitory concentration, 50%) values calculated. 2.5. Real-time reverse transcription-PCR
2. Materials and methods 2.1. Cell culture The Pre-B lineage childhood ALL cell line PreB 697 (recently re-named EU-3 by the original author [28] and also referred to as “697” in cell line repositories) and its GC-resistant descendents, R3F9 and R3D11 were cultured in RPMI 1640 (Invitrogen,
Briefly, total RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit (Qiagen, Crawley, UK) and cDNA synthesis was carried out using the Applied Biosystem High-Capacity cDNA Reverse Transcriptase kit according to the manufacturer’s instructions. Primers and probes for GILZ (Hs00608272 m1), GR (Hs00230813 m1), IB␣ (Hs00153283 m1), Hsp27/HSPB1 (Hs00356629 g1) and TATA-binding protein (TBP) were purchased from Assays-On-Demand (Applied
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Fig. 2. Functional analyses of the GR in GC-sensitive and -resistant cell lines. (A) Basal GR protein expression in the PreB 697 cell lines was assessed by western blotting. (B) GR band intensities were quantified using Fuji image software and normalized to actin. The histogram shows mean ± S.E.M. of three independent experiments. (C) Basal GR mRNA expression in the PreB 697 cell lines as assessed by quantitative real-time PCR. The histogram shows mean ± S.E.M. of three independent experiments. (D) Nuclear translocation of the GR was assessed in all cell lines by exposing cells to a control vehicle (−) or 37 nM dexamethasone (+) and harvested after 4 h, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytosolic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against GR, ␣-tubulin (cytosolic control) and PARP (nuclear control). (E) Similarly, GR translocation in response to dexamethasone (1 M) was assessed in ex vivo-treated primary samples after 4–5-h dosing. (F) Levels of GR:GRE binding in the nuclear fractions of the PreB 697 cell lines after 4 h of 37 nM dexamethasone treatment. The OD450 nm was measured and expressed as the bound GR after 4 h 37 nM dexamethasone treatment minus that of the control vehicle as a percentage of PreB 697 DNA-binding. Columns are the mean and S.E.M. of 3 independent experiments.
Biosystems, Warrington, UK). Samples were assayed in triplicate with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using the TaqMan® universal PCR MasterMix (Applied Biosystems). To quantify mRNA levels the CT method was used with TBP as the endogenous control. 2.6. Western blotting Whole cell lysates were prepared using cell lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS supplemented with protease inhibitor cocktail (Roche, West Sussex, UK). Cytoplasmic and nuclear fractions were prepared as described in [29]. Antibodies supplied by Santa Cruz Biotechnology (Autogen Bioclear) included GR (E-20), BTK (M-138), Hsp27 (F-4), IB␣ (FL), RelA (F-6), p50 (E-10) and PARP (H-250). Also used were c-Rel (Cell Signaling Technology, Danvers, MA, USA), -actin (Calbiochem, San Diego, CA, USA) and ␣-Tubulin (Sigma–Aldrich). Secondary antibodies used were horseradish peroxidase conjugates of either anti-rabbit or anti-mouse IgG (both Dako, Glostrup, Denmark). 2.7. GR-DNA binding ELISA GR-DNA binding affinity in nuclear extracts was quantified by TransAM Transcription Factor ELISA (Active Motif, Carlsbad, CA, USA). Nuclear extracts were prepared as outlined above and 10 g protein was assayed for GR-DNA binding activity according to the manufacturer’s instructions. 2.8. NFB-DNA binding ELISA RelA-DNA binding affinity in nuclear extracts was quantified by Pierce RelA Transcription Factor ELISA (Pierce, Rockford, IL, USA). Nuclear extracts were prepared as outlined above and 10 g protein was assayed
for RelA-DNA binding activity according to the manufacturer’s instructions. 2.9. Statistical analysis Assay data were analysed with univariate General Linear Models (GLM) using SPSS version 15 (SPSS Inc., Chicago, IL). Data were either log-transformed to equalise the variances (assessed with Levene’s homogeneity test within the SPSS GLM procedure) or analysed in the original scale. GLMs with full factorial models were used to analyse the time-course studies with the three cell lines, using time, dexamethasone treatment and cell line as main factors. Linear contrasts were used to compare main factors or treatment interactions. Dose–response data were analysed using the package ‘drc’ [30] in R [31].
3. Results 3.1. GC-sensitivity of the PreB 697 cell line model mirrors that of primary cells GC-sensitivity in vitro was compared in the GC-sensitive, parental cell line, PreB697 and the 2 GC-resistant sub-lines, R3F9 and R3D11 and a cohort of ALL patients at presentation or relapse using an MTS colorimetric assay. These three cell lines differed in GC-sensitivity (ANOVA, F8,33 = 28.576, p < 0.001). The PreB 697 cell line was GC-sensitive with an IC50 of 37 nM (Fig. 1A) while the GCresistant subclones, R3F9 and R3D11, had IC50 values above 10 M.
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Fig. 3. GC-sensitive and GC-resistant cell lines show differential induction of GILZ, GR and IB␣ at the mRNA level after GC-treatment. Effect of dexamethasone on mRNA expression levels of key modulators of NFB activity: GILZ (A), GR (B), IB␣ (C) and Hsp27 (D). PreB 697 cell lines were treated with either a control vehicle (CV) or 37 nM dexamethasone for 0, 6 and 24 h before determining mRNA levels by RQ-RT-PCR. Expression levels were normalized to an endogenous control, TBP, by the Ct method. Fold differences were calculated by comparison with time 0. Bars are mean of three independent experiments; error bars are S.E.M.
These GC-resistant subclones also differed significantly from one another (ANOVA, F4,22 = 3.3991, p = 0.026) and the R3D11 cells were most resistant to GC. Phenotypic stability of the cell lines, with regards to GC-resistance, was assessed by comparing GC-sensitivity at an early (less than 5 passages) or late passage (greater than 15 passage number). Similar dose–response curves were observed between early and late passage number (Fig. 1B). Dose–response curves for patient samples (n = 19) are shown in Fig. 1C. The range of patient IC50 values was very similar to that of the cell lines with the most sensitive, L705, having an IC50 of 41.4 nM compared to those which were inherently GC-resistant with IC50 values of more than 10 M, including three patient samples at relapse. The difference between the most-sensitive and most-resistant patient represents more than 200-fold range in the level of resistance. Similarly, the difference between the most sensitive and most resistant cell line was 241-fold. The similarities in GC-sensitivities between patientderived cells and the PreB 697 cell lines supports the suitability of this model to study GC-resistance in childhood ALL. 3.2. Functional analyses of the GR in the PreB 697 cell line model Previous studies investigating the mechanisms of GC-resistance in cell line models have found mutation or deletion of GR to
be a common cause of steroid insensitivity leading to impaired receptor-ligand interactions. However, mutation or loss of the GR locus is rare in patient samples. Mutational screening of the GR ligand- and DNA-binding domains of the cell lines used in this study has been previously reported [13] but here we extended the screening to include the N-terminal modulatory domain. No somatic mutations were found and the presence of a common SNP in intron D of the GR (−16G/T) confirmed that the cell lines, irrespective of GC-sensitivity, had two wild-type GR genes. Since GC-induced apoptosis is reliant upon adequate nuclear translocation and DNA-binding, we measured GR levels at the mRNA and protein level as well as functionality in terms of ligandinduced nuclear translocation and GRE binding. R3F9 expressed an equivalent level of GR protein to the parental GC-sensitive cell line, while there was a 50% reduction in GR expression in the R3D11 GC-resistant subclone (general linear model (GLM) univariate ANOVA Tukey post hoc test; F2,6 = 6.653, p = 0.030). This was consistent with the mRNA levels, with the R3D11 having a significantly reduced GR mRNA expression (Fig. 2A, B and C, respectively) (ANOVA F2,6 = 11.611, p = 0.009). To assess nuclear translocation, nuclear and cytoplasmic fractions were prepared at time 0 and after treatment with GC and again probed with a GR antibody (Fig. 2D). For all cell lines, the GR was located predominantly in the cytoplasm
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Fig. 4. GC-sensitive and GC-resistant cell lines show differential induction of GR and IB␣ at the protein level after GC-treatment. Time course of GR␣, IB␣, BTK and Hsp27 protein expression in PreB 697, R3F9 and R3D11 after exposure to 37 nM dexamethasone and harvested at the indicated time points. Total protein (15 g) was separated by SDS-PAGE and immunoblotted for GR, IB␣, Hsp27 and actin protein expression. Data shown are representative of three independent experiments. The graphs below the blots show the band intensities as quantified using Fuji image software and normalized to actin. Fold-differences were calculated by comparison with time 0.
of the untreated cells and control vehicle treated cells. After dexamethasone treatment, the GR translocated from the cytoplasm to the nucleus in all cell lines, regardless of GC-sensitive or GC-resistant status. To compare the results from the cell lines to clinical samples, GR nuclear translocation in four patient samples, three at presentation and one at relapse, were similarly studied ex vivo (Fig. 2E). All patient samples exhibited GR nuclear translocation, although levels of nuclear GR were variable. Binding of the GR after dexamethasone treatment to a GRE was assessed in the cell lines using an ELISA-based assay (Fig. 2F) and showed that all cell lines exhibited ligand-induced DNA binding to varying degrees. Basal GR binding was negligible in all cell lines (data not shown). Consistent with GR protein levels, the R3D11 cell line had a significantly reduced level of GR-DNA binding (p = 0.011, two-tailed t-test) while the R3F9 had a lower level of GRE binding which did not reach statistical significance (p = 0.157, two-tailed t-test). 3.3. GC-resistance in the PreB 697 cell line model is associated with a differential induction of the NFB modulators GILZ, GR and IB˛ Several microarray studies on patient samples and cell lines, including the PreB 697 cell line model, have documented differential signature profiles of the negative NFB modulators GR, GILZ and IB␣ and the positive NFB modulators, Hsp27 and BTK, in relation to GC-sensitivity. Thus, we performed RQ-PCR to com-
pare mRNA expression of GILZ, GR, IB␣ and Hsp27 basally and after 6 and 24 h of exposure to dexamethasone compared to control vehicle (Fig. 3). We excluded BTK after finding that all cell lines expressed a truncated form of BTK (Supplementary Fig. 1) which was shown elsewhere to be due to a deletion affecting the kinase domain, thus ruling out BTK from our hypothesis [32]. The response to dexamethasone was compared between cell lines by combining data for 6 and 24 h and showed a significant increase in GILZ expression in the GC-sensitive parental cell line in response to dexamethasone, with a greater than 20-fold increase which differed significantly from the R3D11 cell line (linear contrast, p = 0.014), although the difference between the PreB697 and R3F9 was marginal (p = 0.1). Clearly, both GC-resistant sub clones showed induction of GILZ but to a much lesser extent than the parental cell line (Fig. 3A). GR mRNA was induced in all three cell lines in response to dexamethasone (F1,24 = 59, p < 0.001). The PreB697 cells showed a 3-fold induction of GR mRNA which was significantly greater than in the GC-resistant R3D11 cells (linear contrast, p = 0.002); the difference in GR mRNA induction between PreB697 and R3F9 cells fell just outside the criterion for significance (linear contrast, p = 0.054) (Fig. 3B). For IkB␣ mRNA, there was a significant induction (2-fold or greater) in response to dexamethasone in all cell lines (F1,24 = 64.042, p < 0.001), but no significant differences with respect to cell line or time of treatment (p > 0.05 for main effects and interactions) (Fig. 3C). Messenger RNA levels of Hsp27 did not change significantly in response to treatment with dexam-
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Fig. 5. GC-resistance in the PreB 697 cell lines is not associated with differential NFB nuclear localization. Subcellular fractionation to detect localization of the NFB subunits, RelA, p50 and c-Rel was performed in the PreB 697 (A), R3F9 (B) and R3D11 (C) cell lines. Localization of the NFB subunits was assessed in all cell lines by exposing cells to 37 nM dexamethasone and harvested at various time points up to 48 h, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytoplasmic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against RelA, p50 and c-Rel, ␣-tubulin (cytoplasmic control) and PARP (nuclear control). Similar results were obtained in three separate sets of experiments. (D) Basal levels of RelA-B-binding within the PreB 697 cell lines compared to the multiple myeloma cell line, RPMI 8226. (i) Nuclear lysates from untreated cell lines assayed for NFB binding to -B consensus sequence and imaged using FUJIFILM Luminescent Image Analyzer LAS-3000 camera. (ii) Fold-difference in -B binding relative to PreB 697. Columns represent the mean of three independent experiments; error bars represent the S.E.M.
ethasone in any cell line or time of treatment (GLM, F11,24 = 0.555, p > 0.8) (Fig. 3D). To extend the investigation to include protein expression, all cell lines were exposed to dexamethasone or control vehicle and protein levels were assayed for GR, IB␣ and Hsp 27 over a 24-h time-course. Lack of a commercially available antibody against GILZ prevented analyses of this protein. After dexamethasone treatment, there was a differential induction of proteins seen in the PreB 697 cell lines. Dexamethasone treated samples are shown in Fig. 4 while the vehicle control treated samples are shown in supplementary Fig. 2. Quantification by densitometry revealed a modest increase in GR␣ levels after dexamethasone exposure which was significantly
different to the CV (GLM, F1,10 = 6.202, p = 0.032). Dexamethasone treatment did not significantly affect GR␣ protein induction in the GC-resistant R3F9 (GLM, F1,10 = 1.256, p = 0.289) or the GC-resistant R3D11 cell line (GLM, F1,10 = 2.521, p = 0.143). Dexamethasone had a significant effect on the induction of IB␣ protein in PreB 697 cell line (GLM, F1,10 = 27.976, p = < 0.001) and marginally significant effect in the R3F9 cell line (GLM, F1,10 = 5.357, p = 0.043) but not the R3D11 cell line (GLM, F1,10 = 0.091, p = 0.769). Hsp27 protein levels showed little change in response to dexamethasone exposure over the 24-h time-course in either the GC-sensitive PreB 697, or the GC-resistant R3F9 and R3D11 cell lines (F29,45 = 4.183, p = 0.976).
Fig. 6. Okadaic acid induces nuclear translocation of rel A and p50 in the PreB 697 cell lines. Subcellular fractionation to detect nuclear translocation of the NFB subunits, RelA and p50 in the GC-sensitive cell line, PreB 697 and GC-resistant cell lines, R3F9 and R3D11. Localization of the NFB subunits was assessed in all cell lines by exposing cells to a control vehicle (−) or 0.2 M okadaic acid (+) for 4 h, harvested, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytoplasmic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against RelA, p50, ␣-tubulin (cytoplasmic control) and PARP (nuclear control).
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3.4. Differential induction of negative modulators of NFB in the PreB 697 cell lines does not impact on subcellular localization or functionality The differential upregulation of GR, GILZ and IB␣ in response to GC at the mRNA and protein level in the GC-sensitive PreB 697 cell line compared to that of the GC-resistant cell lines, R3F9 and R3D11, suggest that GC-induced apoptosis may require factors which functionally inhibit NFB. To address the localization of NFB subunits, subcellular fractionation was performed and the immunoblots probed for RelA, p50 and c-Rel. In the untreated cell lines all NFB subunits were localized to the cytosolic fraction (Fig. 5A–C). The subunits remained within the cytosolic fraction over the time course up to, and including, 48 h, irrespective of treatment. Parallel experiments of p65-B binding showed negligible binding of the three cell lines either basally (Fig. 5D) or after GC dosing (data not shown), while the constitutively active, multiple myeloma cell line RPMI 8226, showed significant binding. As a positive control for NFB responsiveness, cell lines were treated with the NFB activator, okadaic acid and nuclear translocation of RelA and p50 subunits occurred (Fig. 6). Thus, these data show that despite having a functional NFB activation pathway, homo- or heterodimers made up of RelA, p50 or c-Rel are sequestered within the cytoplasm in both the GC-sensitive and GC-resistant cell lines, both basally and in response to GC.
4. Discussion Glucocorticoids induce apoptosis in lymphoid cells and consequently have been widely used in the treatment of childhood ALL and in other lymphoid malignancies for many decades. In spite of their clinical importance, the molecular basis by which leukemic cells acquire GC-resistance is poorly defined. Recent studies show that GR deletion/mutation is rare in primary samples and opens up the possibility that teasing apart the mechanisms underlying GC-resistance may allow its pharmacological reversal. In this study we have used an experimental cell line model of GC-resistance in ALL to investigate a subset of gene targets and their impact on the activity of the transcription factor, NFB. Other leukemic cell lines models of GC-resistance have identified impaired functionality of the GR often due to GR mutation, both of which are rare in primary samples [11,12,33]. However, the PreB 697 GC-resistant model used here has 2 copies of wildtype GR. Since the GRs in these cells are capable of undergoing ligand-induced nuclear translocation and GRE-binding, the model recapitulates several features of GC-resistant primary ALL cells and serves as a useful tool for studying the mechanisms underlying GC-resistance. While we clearly show differential induction of the NFB modulators, GILZ and IB␣ in GC-sensitive compared to GC-resistant sub-lines which might be expected to attenuate NFB, there was no affect on NFB nuclear localization or activity. It is possible that GILZ and IB␣ influence GC-induced apoptosis independently of NFB. For example, GILZ has been shown to be a negative regulator of the RAS/RAF/MEK/ERK MAP kinase pathway [34] and the importance of this pathway in GC-induced apoptosis was highlighted by Tanaka et al. in which the use of MEK inhibitors restored GC-sensitivity in GC-resistant leukemic cell lines [35]. In addition, while the IB␣ protein is largely considered to inhibit NFB by sequestration, it has also been shown to be recruited to the promoter of the Notch target gene, hes1, together with HDAC1 and 5, and is involved in its transcriptional repression [36]. GC-resistance was investigated in two PreB697-derived clones, R3F9 and R3D11, in which the levels of GR protein differed. These results indicate that divergent mechanisms may have arisen in the
GC-resistant cell lines after selective pressure in dexamethasonecontaining growth media. These data also confirm that high receptor content does not necessarily correlate with sensitivity. The GC-resistance manifested in R3D11 is likely due to insufficient levels of basal GR protein due to reduced GR transcription and was associated with lower GRE-binding and low target gene expression, compared to the GC-sensitive parent line. A recent finding by Gruber et al., showed that quite modest changes in basal GR protein level could significantly modulate GC-sensitivity in leukemic cells [37]. While transcriptional regulation of the GR is not well understood, a recent study in B-CLL showed that the phosphodiesterase 4 inhibitor, rolipram, can augment GR transcript levels along with glucocorticoid-mediated apoptosis [38]. The GC-resistant cell line, R3F9, failed to upregulate GILZ and IB˛, a commonly used marker of GR-transcriptional activity to the same extent as the parental GC-sensitive cell line and unlike R3D11, R3F9 expressed equivalent basal levels of GR to the GC-sensitive parent, with similar nuclear levels after ligand binding. This is suggestive of repression of GR binding to DNA and indeed GC-induced GR binding appeared lower in the R3F9 compared to the GCsensitive parental line. A number of mechanisms have been shown to influence GR-DNA binding. For example, the GR is subject to posttranslational modification which can alter the trans-activating efficacy, including sumoylation and phosphorylation [39,40]. Furthermore, GC-resistant ALL primary samples have been associated with a decreased expression of subunits of the SWI/SNF chromatinremodeling complex required for GR transcriptional activity [41]. This is the first study to address the role of the transcription factor, NFB, in glucocorticoid resistance in a leukemic cell line model and suggests that despite differential induction of NFB modulators, NFB does not underlie the GC-resistant phenotype. Further investigation of the impact of the GC-sensitive associated proteins on other survival pathways, such as the RAS-RAF-MEK-ERK pathway, is warranted. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements The authors gratefully acknowledge Leukaemia Research for funding this study and thank Reinhard Kofler for providing the cell lines. Contributions. JI, CR and AH initially conceived the study and LN performed all experiments, analysed data and contributed to the study design. LN and JI drafted the article and all authors critically appraised and approved the final version. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2009.12.014. References [1] Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq C, Yamamoto KR. Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci USA 2004;101:15603–8. [2] Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA 1994;91:752–6. [3] Herr I, Gassler N, Friess H, Buchler MW. Regulation of differential pro- and anti-apoptotic signaling by glucocorticoids. Apoptosis 2007;12:271–91. [4] Riehm H, Reiter A, Schrappe M, Berthold F, Dopfer R, Gerein V, et al. Corticosteroid-dependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83). Klin Padiatr 1987;199:151–60.
L. Nicholson et al. / Leukemia Research 34 (2010) 1366–1373 [5] Reiter A, Schrappe M, Ludwig WD, Hiddemann W, Sauter S, Henze G, et al. Chemotherapy in 998 unselected childhood acute lymphoblastic leukemia patients. Results and conclusions of the multicenter trial ALL-BFM 86. Blood 1994;84:3122–33. [6] Schrappe M, Reiter A, Zimmermann M, Harbott J, Ludwig WD, Henze G, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster. Leukemia 2000;14:2205–22. [7] Klumper E, Pieters R, Veerman A, Huismans D, Loonen A, Hahlen K, et al. In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia. Blood 1995;86:3861–8. [8] Kaspers GJ, Wijnands JJ, Hartmann R, Huismans L, Loonen AH, Stackelberg A, et al. Immunophenotypic cell lineage and in vitro cellular drug resistance in childhood relapsed acute lymphoblastic leukaemia. Eur J Cancer 2005;41:1300–3. [9] Kofler R, Schmidt S, Kofler A, Ausserlechner MJ. Resistance to glucocorticoidinduced apoptosis in lymphoblastic leukemia. J Endocrinol 2003;178:19–27. [10] Haarman EG, Kaspers GJ, Veerman AJ. Glucocorticoid resistance in childhood leukaemia: mechanisms and modulation. Br J Haematol 2003;120:919–29. [11] Irving JA, Minto L, Bailey S, Hall AG. Loss of heterozygosity and somatic mutations of the glucocorticoid receptor gene are rarely found at relapse in pediatric acute lymphoblastic leukemia but may occur in a subpopulation early in the disease course. Cancer Res 2005;65:9712–8. [12] Tissing WJ, Meijerink JP, den Boer ML, Brinkhof B, van Rossum EF, van Wering ER, et al. Genetic variations in the glucocorticoid receptor gene are not related to glucocorticoid resistance in childhood acute lymphoblastic leukemia. Clin Cancer Res 2005;11:6050–6. [13] Schmidt S, Irving JA, Minto L, Matheson E, Nicholson L, Ploner A, et al. Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at the level of the glucocorticoid receptor. FASEB J 2006;20:2600–2. [14] Matheson EC, Hall AG. Assessment of mismatch repair function in leukaemic cell lines and blasts from children with acute lymphoblastic leukaemia. Carcinogenesis 2003;24:31–8. [15] Chauhan D, Auclair D, Robinson EK, Hideshima T, Li G, Podar K, et al. Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays. Oncogene 2002;21:1346–58. [16] Miller AL, Komak S, Webb MS, Leiter EH, Thompson EB. Gene expression profiling of leukemic cells and primary thymocytes predicts a signature for apoptotic sensitivity to glucocorticoids. Cancer Cell Int 2007;7:18. [17] Schmidt S, Rainer J, Riml S, Ploner C, Jesacher S, Achmuller C, et al. Identification of glucocorticoid-response genes in children with acute lymphoblastic leukemia. Blood 2006;107:2061–9. [18] Caldenhoven E, Liden J, Wissink S, Van de Stolpe A, Raaijmakers J, Koenderman L, et al. Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. Mol Endocrinol 1995;9:401–12. [19] Ayroldi E, Migliorati G, Bruscoli S, Marchetti C, Zollo O, Cannarile L, et al. Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB. Blood 2001;98:743–53. [20] Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 2007;8:49–62. [21] Novac N, Baus D, Dostert A, Heinzel T. Competition between glucocorticoid receptor and NFkappaB for control of the human FasL promoter. FASEB J 2006;20:1074–81. [22] Sheppard KA, Phelps KM, Williams AJ, Thanos D, Glass CK, Rosenfeld MG, et al. Nuclear integration of glucocorticoid receptor and nuclear factor-kappaB signaling by CREB-binding protein and steroid receptor coactivator-1. J Biol Chem 1998;273:29291–4.
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[23] Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 1995;270:286–90. [24] Scheinman RI, Cogswell PC, Lofquist AK, Baldwin Jr AS. Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science 1995;270:283–6. [25] Petro JB, Rahman SM, Ballard DW, Khan WN. Bruton’s tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement. J Exp Med 2000;191:1745–54. [26] Parcellier A, Schmitt E, Gurbuxani S, Seigneurin-Berny D, Pance A, Chantome A, et al. HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation. Mol Cell Biol 2003;23:5790–802. [27] Kordes U, Krappmann D, Heissmeyer V, Ludwig WD, Scheidereit C. Transcription factor NF-kappaB is constitutively activated in acute lymphoblastic leukemia cells. Leukemia 2000;14:399–402. [28] Zhou M, Gu L, Li F, Zhu Y, Woods WG, Findley HW. DNA damage induces a novel p53-survivin signaling pathway regulating cell cycle and apoptosis in acute lymphoblastic leukemia cells. J Pharmacol Exp Ther 2002;303: 124–31. [29] Olnes MI, Kurl RN. Isolation of nuclear extracts from fragile cells: a simplified procedure applied to thymocytes. Biotechniques 1994;17:828–9. [30] Ritz C, Streibig J. Bioassay analysis using R. J Stat Softw 2005;12. [31] Team RDC. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2008. [32] Feldhahn N, Rio P, Soh BN, Liedtke S, Sprangers M, Klein F, et al. Deficiency of Bruton’s tyrosine kinase in B cell precursor leukemia cells. Proc Natl Acad Sci USA 2005;102:13266–71. [33] Bachmann PS, Gorman R, Papa RA, Bardell JE, Ford J, Kees UR, et al. Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia. Cancer Res 2007;67:4482–90. [34] Ayroldi E, Zollo O, Macchiarulo A, Di Marco B, Marchetti C, Riccardi C. Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signalregulated kinase pathway by binding to Raf-1. Mol Cell Biol 2002;22: 7929–41. [35] Tanaka T, Okabe T, Gondo S, Fukuda M, Yamamoto M, Umemura T, et al. Modification of glucocorticoid sensitivity by MAP kinase signaling pathways in glucocorticoid-induced T-cell apoptosis. Exp Hematol 2006;34: 1542–52. [36] Aguilera C, Hoya-Arias R, Haegeman G, Espinosa L, Bigas A. Recruitment of IkappaBalpha to the hes1 promoter is associated with transcriptional repression. Proc Natl Acad Sci USA 2004;101:16537–42. [37] Gruber G, Carlet M, Turtscher E, Meister B, Irving JA, Ploner C, et al. Levels of glucocorticoid receptor and its ligand determine sensitivity and kinetics of glucocorticoid-induced leukemia apoptosis. Leukemia 2009;23:820–3. [38] Meyers JA, Taverna J, Chaves J, Makkinje A, Lerner A. Phosphodiesterase 4 inhibitors augment levels of glucocorticoid receptor in B cell chronic lymphocytic leukemia but not in normal circulating hematopoietic cells. Clin Cancer Res 2007;13:4920–7. [39] Le Drean Y, Mincheneau N, Le Goff P, Michel D. Potentiation of glucocorticoid receptor transcriptional activity by sumoylation. Endocrinology 2002;143:3482–9. [40] Bodwell JE, Webster JC, Jewell CM, Cidlowski JA, Hu JM, Munck A. Glucocorticoid receptor phosphorylation: overview, function and cell cycle-dependence. J Steroid Biochem Mol Biol 1998;65:91–9. [41] Pottier N, Yang W, Assem M, Panetta JC, Pei D, Paugh SW, et al. The SWI/SNF chromatin-remodeling complex and glucocorticoid resistance in acute lymphoblastic leukemia. J Natl Cancer Inst 2008;100:1792–803.
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NFB modulators in a model of glucocorticoid resistant, childhood acute lymphoblastic leukemia Lindsay Nicholson, Andrew G. Hall, Christopher P. Redfern, Julie Irving ∗ Northern Institute for Cancer Research, Newcastle University, Paul O’Gorman Building, Framlington Place, Newcastle upon Tyne, Tyne and Wear, NE2 4HH, UK
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Article history: Received 23 October 2009 Received in revised form 19 December 2009 Accepted 19 December 2009 Available online 27 January 2010 Keywords: Glucocorticoid resistance Childhood acute lymphoblastic leukaemia NFB
a b s t r a c t Glucocorticoids (GCs) are pivotal agents in the treatment of childhood acute lymphoblastic leukaemia (ALL) but the molecular basis of GC-resistance remains unclear. Expression-array studies have shown that commonly upregulated genes associated with GC-sensitivity include GR, glucocorticoid-induced leucine zipper (GILZ) and IB␣, which all negatively interact with components of the pro-survival NFB pathway and therefore may be critical determinants of GC-sensitivity. We have investigated these regulators and their effect on NFB activity in GC-resistant descendents of the B-lineage ALL cell line, PreB 697. We show that while differential up regulation of the modulators (GILZ, GR and IB␣) was demonstrated in GCsensitive compared to GC-resistant sub-lines, this was not coupled with altered nuclear translocation or functionality of the RelA, p50 or c-Rel subunits of NFB. Thus, GC-resistance in the PreB 697 cell line model is not mediated by NFB, however further investigation of the impact of these GC-sensitive associated proteins on other survival pathways, such as the RAS-RAF-MEK-ERK pathway, is warranted. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Glucocorticoids are central to the treatment regime of all lymphoid malignancies, including childhood acute lymphoblastic leukaemia (ALL), due to their ability to induce apoptosis in immature lymphoid cells. The effects are mediated by the glucocorticoid receptor (GR, also known as NR3C1), a ligand-activated transcription factor belonging to the nuclear receptor superfamily. In an unstimulated cell, the GR is located within the cytoplasm bound to a heteromeric complex of heat shock proteins and immunophilins, upon ligand binding the GR undergoes nuclear translocation, dimerizes and can influence gene expression in several ways. The GC–GR complex can bind to DNA and trigger transactivation or transrepression of glucocorticoid response elements (GRE) in target genes [1]. Additionally, the GC–GR complex can indirectly influence gene transcription by protein–protein interactions with other transcription factors, for example, nuclear factor-B (NFB) and AP-1, repressing transcription of their target genes [2]. It is this complex interplay that results in changes in gene expression and ultimately leads to caspase activation and apoptosis in GC-sensitive cells [3]. In childhood ALL, glucocorticoid sensitivity in vitro and in vivo is a significant prognostic indicator and in three consecutive BerlinFrankfurt-Münster (BFM) trials patients responding well to a short mono-therapy GC window were shown to have a better outcome than poor responders [4–6]. Furthermore, leukemic cells of patients
∗ Corresponding author. Tel.: +44 191 246 4369; fax: +44 191 246 4301. E-mail address: [email protected] (J. Irving). 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.12.014
with relapsed ALL are up to 300-fold more resistant to GC in vitro than those taken at diagnosis [7,8] and with relapse rates at 20%, GC-resistance remains a continuing problem. However, despite the inclusion of GCs in treatment protocols for over 50 years, the mechanisms behind defining GC-sensitive or resistant childhood ALL remains elusive [9,10]. Primary patient cells are the obvious choice with which to study GC-resistance in childhood ALL, but spontaneous apoptosis of leukemic blasts in prolonged cell culture and limited amounts of material have made cell line models a valuable alternative. Commonly used GC-resistant, cell line derivatives, such as those of CCRF-CEM and Jurkat, demonstrate loss/mutation of the GR as an acquired GC-resistance mechanism but these events are rarely found in primary ALL cells [11,12] and cannot account for most cases of GC-insensitivity. This disparity is probably due to the mismatch repair deficient status of many ALL cell lines, including CCRF-CEM, since deficiency is associated with an increased basal mutation rate and thus a greater likelihood of acquiring GR mutations under GC selection pressure [13]. In contrast, GC-resistant descendants of a mismatch repair proficient cell lines, PreB 697, predominantly retained two wild-type GR alleles. Again, mismatch repair deficiency, a predominate feature of ALL cell lines, is rarely found in primary ALL samples [14]. A number of studies have used gene expression arrays to try to identify key genes that are transcriptionally regulated in response to GC and may be determinants of GC-sensitivity. Upregulated genes, consistently associated with GC-sensitivity in several studies, included the GR itself, glucocorticoid-induced leucine zipper (GILZ, also known as TSC22D3) and IB␣, (also known as NFB1)
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Fig. 1. Response of PreB 697 cell lines and clinical samples to dexamethasone exposure in vitro. Cell viability was assessed using the MTS assay after a 96-h drug exposure. The viable cell number at each drug concentration was calculated relative to vehicle-control treated cells. Cell line data are the mean ± S.E.M. of at least three independent experiments, patient data are mean ± S.E.M. of at least three wells from a single experiment. (A) GC-sensitivity in the parental PreB 697 and two descendents, R3F9 and R3D11. (B) Phenotypic stability of GC-resistance in the PreB 697 cell lines after early (<5 passages) and late (>15 passages) passage number. (C) GC-sensitivity in a panel of ALL patient samples taken at presentation and relapse (R denotes relapse).
[15–17] all of which have been shown to negatively impact with components of the pro-survival NFB family [18–20]. In unstimulated cells, NFB is sequestered within the cytoplasm, bound to its inhibitory proteins, IB, which block its nuclear translocation. Upon stimulation, for example by TNF␣ exposure, a cascade of signalling events occur, eventually culminating in proteosomal degradation of IB allowing NFB to translocate to the nucleus where it can regulate gene transcription. GR can directly antagonise NFB activity by competing for co-activators or promoter binding [2,21,22] and indirectly by upregulating the synthesis of IkB [23,24]. Further evidence implicating NFB activity in modulating GC-sensitivity comes from a study of paired GC-sensitive and GC-resistant multiple myeloma cell lines in which upregulation of Bruton’s Tyrosine Kinase (BTK) and Hsp27 was associated with GC-resistance [15]. Both of these proteins have been implicated in activating NFB [25,26]. In addition, constitutively activated NF-kappaB has been identified in cALL [27]. The strong correlation between response to GC mono-therapy and clinical outcome is intriguing given that subsequent therapy involves exposure to 6 or 7 other drugs with diverse mechanisms of actions. However, the unifying end response of any chemotherapeutic agent is to trigger cell death. This implies that GC-resistance may be a reflection of the intrinsic propensity of the blasts to undergo apoptosis and that resistance to GC is a more generalised mechanism, affecting response to other therapeutic agents i.e. an increased apoptotic threshold, as might be afforded by variations in NFB signalling. Thus, in this study, we have used the PreB 697 cell lines and two GC-resistant descendents, to investigate GC-related modulators of NFB, their impact on its activity and their relevance to GC-induced apoptosis.
Paisley, UK) supplemented with 10% foetal bovine serum (Invitrogen) at 37 ◦ C and 5% carbon dioxide. The PreB 697, R3F9 and R3D11 cell lines were kindly donated by R. Kofler [13]. The multiple myeloma cell line, RPMI 8226, was purchased from ECACC and cultured in RPMI 1640 supplemented with 10% foetal bovine serum. All cell lines were routinely tested for mycoplasma contamination using MycoAlert® (Lonza, Basel, Switzerland). 2.2. Patient samples Mononuclear cells from bone marrow from untreated children presenting with ALL at the Royal Victoria Infirmary, Newcastle upon Tyne, UK, were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). The cohort consisted of 14 patients (age range, 1.2–18 years), 7 males, 7 females and were predominantly B-lineage (n = 13). Regional ethical committee approval was obtained for the study (reference numbers 2002/111 and 07/H0906). 2.3. Mutational screening GR exon 2 and flanking intronic sequences were amplified from 100–200 ng genomic DNA from all cell lines and were mutationally screened by denaturing HPLC and direct sequencing. Forward and reverse primer sequences, annealing temperatures and denaturing HPLC parameters were performed as previously described [11]. 2.4. In vitro drug sensitivity assays Cell lines or patient cells were plated out in triplicate at 2 × 104 or 2 × 105 , respectively, per well into 96-well plates. Cells were treated with either dexamethasone (Sigma–Aldrich, Dorset, UK) to a range of final concentrations (10−9 to 10−5 M) or control vehicle. Following a 96-h drug exposure, cytotoxicity was assessed using the CellTiter 96 Aqueous One kit (Promega, Southampton, UK), which assesses the capacity of cells to reduce formazan and thus is a measure of metabolically active cells. The resulting absorbances were averaged and expressed as a percentage of the control vehicle. Survival curves were plotted using GraphPad Prism software and IC50 (inhibitory concentration, 50%) values calculated. 2.5. Real-time reverse transcription-PCR
2. Materials and methods 2.1. Cell culture The Pre-B lineage childhood ALL cell line PreB 697 (recently re-named EU-3 by the original author [28] and also referred to as “697” in cell line repositories) and its GC-resistant descendents, R3F9 and R3D11 were cultured in RPMI 1640 (Invitrogen,
Briefly, total RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit (Qiagen, Crawley, UK) and cDNA synthesis was carried out using the Applied Biosystem High-Capacity cDNA Reverse Transcriptase kit according to the manufacturer’s instructions. Primers and probes for GILZ (Hs00608272 m1), GR (Hs00230813 m1), IB␣ (Hs00153283 m1), Hsp27/HSPB1 (Hs00356629 g1) and TATA-binding protein (TBP) were purchased from Assays-On-Demand (Applied
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Fig. 2. Functional analyses of the GR in GC-sensitive and -resistant cell lines. (A) Basal GR protein expression in the PreB 697 cell lines was assessed by western blotting. (B) GR band intensities were quantified using Fuji image software and normalized to actin. The histogram shows mean ± S.E.M. of three independent experiments. (C) Basal GR mRNA expression in the PreB 697 cell lines as assessed by quantitative real-time PCR. The histogram shows mean ± S.E.M. of three independent experiments. (D) Nuclear translocation of the GR was assessed in all cell lines by exposing cells to a control vehicle (−) or 37 nM dexamethasone (+) and harvested after 4 h, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytosolic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against GR, ␣-tubulin (cytosolic control) and PARP (nuclear control). (E) Similarly, GR translocation in response to dexamethasone (1 M) was assessed in ex vivo-treated primary samples after 4–5-h dosing. (F) Levels of GR:GRE binding in the nuclear fractions of the PreB 697 cell lines after 4 h of 37 nM dexamethasone treatment. The OD450 nm was measured and expressed as the bound GR after 4 h 37 nM dexamethasone treatment minus that of the control vehicle as a percentage of PreB 697 DNA-binding. Columns are the mean and S.E.M. of 3 independent experiments.
Biosystems, Warrington, UK). Samples were assayed in triplicate with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using the TaqMan® universal PCR MasterMix (Applied Biosystems). To quantify mRNA levels the CT method was used with TBP as the endogenous control. 2.6. Western blotting Whole cell lysates were prepared using cell lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS supplemented with protease inhibitor cocktail (Roche, West Sussex, UK). Cytoplasmic and nuclear fractions were prepared as described in [29]. Antibodies supplied by Santa Cruz Biotechnology (Autogen Bioclear) included GR (E-20), BTK (M-138), Hsp27 (F-4), IB␣ (FL), RelA (F-6), p50 (E-10) and PARP (H-250). Also used were c-Rel (Cell Signaling Technology, Danvers, MA, USA), -actin (Calbiochem, San Diego, CA, USA) and ␣-Tubulin (Sigma–Aldrich). Secondary antibodies used were horseradish peroxidase conjugates of either anti-rabbit or anti-mouse IgG (both Dako, Glostrup, Denmark). 2.7. GR-DNA binding ELISA GR-DNA binding affinity in nuclear extracts was quantified by TransAM Transcription Factor ELISA (Active Motif, Carlsbad, CA, USA). Nuclear extracts were prepared as outlined above and 10 g protein was assayed for GR-DNA binding activity according to the manufacturer’s instructions. 2.8. NFB-DNA binding ELISA RelA-DNA binding affinity in nuclear extracts was quantified by Pierce RelA Transcription Factor ELISA (Pierce, Rockford, IL, USA). Nuclear extracts were prepared as outlined above and 10 g protein was assayed
for RelA-DNA binding activity according to the manufacturer’s instructions. 2.9. Statistical analysis Assay data were analysed with univariate General Linear Models (GLM) using SPSS version 15 (SPSS Inc., Chicago, IL). Data were either log-transformed to equalise the variances (assessed with Levene’s homogeneity test within the SPSS GLM procedure) or analysed in the original scale. GLMs with full factorial models were used to analyse the time-course studies with the three cell lines, using time, dexamethasone treatment and cell line as main factors. Linear contrasts were used to compare main factors or treatment interactions. Dose–response data were analysed using the package ‘drc’ [30] in R [31].
3. Results 3.1. GC-sensitivity of the PreB 697 cell line model mirrors that of primary cells GC-sensitivity in vitro was compared in the GC-sensitive, parental cell line, PreB697 and the 2 GC-resistant sub-lines, R3F9 and R3D11 and a cohort of ALL patients at presentation or relapse using an MTS colorimetric assay. These three cell lines differed in GC-sensitivity (ANOVA, F8,33 = 28.576, p < 0.001). The PreB 697 cell line was GC-sensitive with an IC50 of 37 nM (Fig. 1A) while the GCresistant subclones, R3F9 and R3D11, had IC50 values above 10 M.
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Fig. 3. GC-sensitive and GC-resistant cell lines show differential induction of GILZ, GR and IB␣ at the mRNA level after GC-treatment. Effect of dexamethasone on mRNA expression levels of key modulators of NFB activity: GILZ (A), GR (B), IB␣ (C) and Hsp27 (D). PreB 697 cell lines were treated with either a control vehicle (CV) or 37 nM dexamethasone for 0, 6 and 24 h before determining mRNA levels by RQ-RT-PCR. Expression levels were normalized to an endogenous control, TBP, by the Ct method. Fold differences were calculated by comparison with time 0. Bars are mean of three independent experiments; error bars are S.E.M.
These GC-resistant subclones also differed significantly from one another (ANOVA, F4,22 = 3.3991, p = 0.026) and the R3D11 cells were most resistant to GC. Phenotypic stability of the cell lines, with regards to GC-resistance, was assessed by comparing GC-sensitivity at an early (less than 5 passages) or late passage (greater than 15 passage number). Similar dose–response curves were observed between early and late passage number (Fig. 1B). Dose–response curves for patient samples (n = 19) are shown in Fig. 1C. The range of patient IC50 values was very similar to that of the cell lines with the most sensitive, L705, having an IC50 of 41.4 nM compared to those which were inherently GC-resistant with IC50 values of more than 10 M, including three patient samples at relapse. The difference between the most-sensitive and most-resistant patient represents more than 200-fold range in the level of resistance. Similarly, the difference between the most sensitive and most resistant cell line was 241-fold. The similarities in GC-sensitivities between patientderived cells and the PreB 697 cell lines supports the suitability of this model to study GC-resistance in childhood ALL. 3.2. Functional analyses of the GR in the PreB 697 cell line model Previous studies investigating the mechanisms of GC-resistance in cell line models have found mutation or deletion of GR to
be a common cause of steroid insensitivity leading to impaired receptor-ligand interactions. However, mutation or loss of the GR locus is rare in patient samples. Mutational screening of the GR ligand- and DNA-binding domains of the cell lines used in this study has been previously reported [13] but here we extended the screening to include the N-terminal modulatory domain. No somatic mutations were found and the presence of a common SNP in intron D of the GR (−16G/T) confirmed that the cell lines, irrespective of GC-sensitivity, had two wild-type GR genes. Since GC-induced apoptosis is reliant upon adequate nuclear translocation and DNA-binding, we measured GR levels at the mRNA and protein level as well as functionality in terms of ligandinduced nuclear translocation and GRE binding. R3F9 expressed an equivalent level of GR protein to the parental GC-sensitive cell line, while there was a 50% reduction in GR expression in the R3D11 GC-resistant subclone (general linear model (GLM) univariate ANOVA Tukey post hoc test; F2,6 = 6.653, p = 0.030). This was consistent with the mRNA levels, with the R3D11 having a significantly reduced GR mRNA expression (Fig. 2A, B and C, respectively) (ANOVA F2,6 = 11.611, p = 0.009). To assess nuclear translocation, nuclear and cytoplasmic fractions were prepared at time 0 and after treatment with GC and again probed with a GR antibody (Fig. 2D). For all cell lines, the GR was located predominantly in the cytoplasm
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Fig. 4. GC-sensitive and GC-resistant cell lines show differential induction of GR and IB␣ at the protein level after GC-treatment. Time course of GR␣, IB␣, BTK and Hsp27 protein expression in PreB 697, R3F9 and R3D11 after exposure to 37 nM dexamethasone and harvested at the indicated time points. Total protein (15 g) was separated by SDS-PAGE and immunoblotted for GR, IB␣, Hsp27 and actin protein expression. Data shown are representative of three independent experiments. The graphs below the blots show the band intensities as quantified using Fuji image software and normalized to actin. Fold-differences were calculated by comparison with time 0.
of the untreated cells and control vehicle treated cells. After dexamethasone treatment, the GR translocated from the cytoplasm to the nucleus in all cell lines, regardless of GC-sensitive or GC-resistant status. To compare the results from the cell lines to clinical samples, GR nuclear translocation in four patient samples, three at presentation and one at relapse, were similarly studied ex vivo (Fig. 2E). All patient samples exhibited GR nuclear translocation, although levels of nuclear GR were variable. Binding of the GR after dexamethasone treatment to a GRE was assessed in the cell lines using an ELISA-based assay (Fig. 2F) and showed that all cell lines exhibited ligand-induced DNA binding to varying degrees. Basal GR binding was negligible in all cell lines (data not shown). Consistent with GR protein levels, the R3D11 cell line had a significantly reduced level of GR-DNA binding (p = 0.011, two-tailed t-test) while the R3F9 had a lower level of GRE binding which did not reach statistical significance (p = 0.157, two-tailed t-test). 3.3. GC-resistance in the PreB 697 cell line model is associated with a differential induction of the NFB modulators GILZ, GR and IB˛ Several microarray studies on patient samples and cell lines, including the PreB 697 cell line model, have documented differential signature profiles of the negative NFB modulators GR, GILZ and IB␣ and the positive NFB modulators, Hsp27 and BTK, in relation to GC-sensitivity. Thus, we performed RQ-PCR to com-
pare mRNA expression of GILZ, GR, IB␣ and Hsp27 basally and after 6 and 24 h of exposure to dexamethasone compared to control vehicle (Fig. 3). We excluded BTK after finding that all cell lines expressed a truncated form of BTK (Supplementary Fig. 1) which was shown elsewhere to be due to a deletion affecting the kinase domain, thus ruling out BTK from our hypothesis [32]. The response to dexamethasone was compared between cell lines by combining data for 6 and 24 h and showed a significant increase in GILZ expression in the GC-sensitive parental cell line in response to dexamethasone, with a greater than 20-fold increase which differed significantly from the R3D11 cell line (linear contrast, p = 0.014), although the difference between the PreB697 and R3F9 was marginal (p = 0.1). Clearly, both GC-resistant sub clones showed induction of GILZ but to a much lesser extent than the parental cell line (Fig. 3A). GR mRNA was induced in all three cell lines in response to dexamethasone (F1,24 = 59, p < 0.001). The PreB697 cells showed a 3-fold induction of GR mRNA which was significantly greater than in the GC-resistant R3D11 cells (linear contrast, p = 0.002); the difference in GR mRNA induction between PreB697 and R3F9 cells fell just outside the criterion for significance (linear contrast, p = 0.054) (Fig. 3B). For IkB␣ mRNA, there was a significant induction (2-fold or greater) in response to dexamethasone in all cell lines (F1,24 = 64.042, p < 0.001), but no significant differences with respect to cell line or time of treatment (p > 0.05 for main effects and interactions) (Fig. 3C). Messenger RNA levels of Hsp27 did not change significantly in response to treatment with dexam-
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Fig. 5. GC-resistance in the PreB 697 cell lines is not associated with differential NFB nuclear localization. Subcellular fractionation to detect localization of the NFB subunits, RelA, p50 and c-Rel was performed in the PreB 697 (A), R3F9 (B) and R3D11 (C) cell lines. Localization of the NFB subunits was assessed in all cell lines by exposing cells to 37 nM dexamethasone and harvested at various time points up to 48 h, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytoplasmic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against RelA, p50 and c-Rel, ␣-tubulin (cytoplasmic control) and PARP (nuclear control). Similar results were obtained in three separate sets of experiments. (D) Basal levels of RelA-B-binding within the PreB 697 cell lines compared to the multiple myeloma cell line, RPMI 8226. (i) Nuclear lysates from untreated cell lines assayed for NFB binding to -B consensus sequence and imaged using FUJIFILM Luminescent Image Analyzer LAS-3000 camera. (ii) Fold-difference in -B binding relative to PreB 697. Columns represent the mean of three independent experiments; error bars represent the S.E.M.
ethasone in any cell line or time of treatment (GLM, F11,24 = 0.555, p > 0.8) (Fig. 3D). To extend the investigation to include protein expression, all cell lines were exposed to dexamethasone or control vehicle and protein levels were assayed for GR, IB␣ and Hsp 27 over a 24-h time-course. Lack of a commercially available antibody against GILZ prevented analyses of this protein. After dexamethasone treatment, there was a differential induction of proteins seen in the PreB 697 cell lines. Dexamethasone treated samples are shown in Fig. 4 while the vehicle control treated samples are shown in supplementary Fig. 2. Quantification by densitometry revealed a modest increase in GR␣ levels after dexamethasone exposure which was significantly
different to the CV (GLM, F1,10 = 6.202, p = 0.032). Dexamethasone treatment did not significantly affect GR␣ protein induction in the GC-resistant R3F9 (GLM, F1,10 = 1.256, p = 0.289) or the GC-resistant R3D11 cell line (GLM, F1,10 = 2.521, p = 0.143). Dexamethasone had a significant effect on the induction of IB␣ protein in PreB 697 cell line (GLM, F1,10 = 27.976, p = < 0.001) and marginally significant effect in the R3F9 cell line (GLM, F1,10 = 5.357, p = 0.043) but not the R3D11 cell line (GLM, F1,10 = 0.091, p = 0.769). Hsp27 protein levels showed little change in response to dexamethasone exposure over the 24-h time-course in either the GC-sensitive PreB 697, or the GC-resistant R3F9 and R3D11 cell lines (F29,45 = 4.183, p = 0.976).
Fig. 6. Okadaic acid induces nuclear translocation of rel A and p50 in the PreB 697 cell lines. Subcellular fractionation to detect nuclear translocation of the NFB subunits, RelA and p50 in the GC-sensitive cell line, PreB 697 and GC-resistant cell lines, R3F9 and R3D11. Localization of the NFB subunits was assessed in all cell lines by exposing cells to a control vehicle (−) or 0.2 M okadaic acid (+) for 4 h, harvested, followed by subcellular fractionation and immunoblot analysis. Equal amounts of protein (15 g) from cytoplasmic (C) and nuclear (N) fractions were separated by SDS-PAGE and membranes probed using antibodies against RelA, p50, ␣-tubulin (cytoplasmic control) and PARP (nuclear control).
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3.4. Differential induction of negative modulators of NFB in the PreB 697 cell lines does not impact on subcellular localization or functionality The differential upregulation of GR, GILZ and IB␣ in response to GC at the mRNA and protein level in the GC-sensitive PreB 697 cell line compared to that of the GC-resistant cell lines, R3F9 and R3D11, suggest that GC-induced apoptosis may require factors which functionally inhibit NFB. To address the localization of NFB subunits, subcellular fractionation was performed and the immunoblots probed for RelA, p50 and c-Rel. In the untreated cell lines all NFB subunits were localized to the cytosolic fraction (Fig. 5A–C). The subunits remained within the cytosolic fraction over the time course up to, and including, 48 h, irrespective of treatment. Parallel experiments of p65-B binding showed negligible binding of the three cell lines either basally (Fig. 5D) or after GC dosing (data not shown), while the constitutively active, multiple myeloma cell line RPMI 8226, showed significant binding. As a positive control for NFB responsiveness, cell lines were treated with the NFB activator, okadaic acid and nuclear translocation of RelA and p50 subunits occurred (Fig. 6). Thus, these data show that despite having a functional NFB activation pathway, homo- or heterodimers made up of RelA, p50 or c-Rel are sequestered within the cytoplasm in both the GC-sensitive and GC-resistant cell lines, both basally and in response to GC.
4. Discussion Glucocorticoids induce apoptosis in lymphoid cells and consequently have been widely used in the treatment of childhood ALL and in other lymphoid malignancies for many decades. In spite of their clinical importance, the molecular basis by which leukemic cells acquire GC-resistance is poorly defined. Recent studies show that GR deletion/mutation is rare in primary samples and opens up the possibility that teasing apart the mechanisms underlying GC-resistance may allow its pharmacological reversal. In this study we have used an experimental cell line model of GC-resistance in ALL to investigate a subset of gene targets and their impact on the activity of the transcription factor, NFB. Other leukemic cell lines models of GC-resistance have identified impaired functionality of the GR often due to GR mutation, both of which are rare in primary samples [11,12,33]. However, the PreB 697 GC-resistant model used here has 2 copies of wildtype GR. Since the GRs in these cells are capable of undergoing ligand-induced nuclear translocation and GRE-binding, the model recapitulates several features of GC-resistant primary ALL cells and serves as a useful tool for studying the mechanisms underlying GC-resistance. While we clearly show differential induction of the NFB modulators, GILZ and IB␣ in GC-sensitive compared to GC-resistant sub-lines which might be expected to attenuate NFB, there was no affect on NFB nuclear localization or activity. It is possible that GILZ and IB␣ influence GC-induced apoptosis independently of NFB. For example, GILZ has been shown to be a negative regulator of the RAS/RAF/MEK/ERK MAP kinase pathway [34] and the importance of this pathway in GC-induced apoptosis was highlighted by Tanaka et al. in which the use of MEK inhibitors restored GC-sensitivity in GC-resistant leukemic cell lines [35]. In addition, while the IB␣ protein is largely considered to inhibit NFB by sequestration, it has also been shown to be recruited to the promoter of the Notch target gene, hes1, together with HDAC1 and 5, and is involved in its transcriptional repression [36]. GC-resistance was investigated in two PreB697-derived clones, R3F9 and R3D11, in which the levels of GR protein differed. These results indicate that divergent mechanisms may have arisen in the
GC-resistant cell lines after selective pressure in dexamethasonecontaining growth media. These data also confirm that high receptor content does not necessarily correlate with sensitivity. The GC-resistance manifested in R3D11 is likely due to insufficient levels of basal GR protein due to reduced GR transcription and was associated with lower GRE-binding and low target gene expression, compared to the GC-sensitive parent line. A recent finding by Gruber et al., showed that quite modest changes in basal GR protein level could significantly modulate GC-sensitivity in leukemic cells [37]. While transcriptional regulation of the GR is not well understood, a recent study in B-CLL showed that the phosphodiesterase 4 inhibitor, rolipram, can augment GR transcript levels along with glucocorticoid-mediated apoptosis [38]. The GC-resistant cell line, R3F9, failed to upregulate GILZ and IB˛, a commonly used marker of GR-transcriptional activity to the same extent as the parental GC-sensitive cell line and unlike R3D11, R3F9 expressed equivalent basal levels of GR to the GC-sensitive parent, with similar nuclear levels after ligand binding. This is suggestive of repression of GR binding to DNA and indeed GC-induced GR binding appeared lower in the R3F9 compared to the GCsensitive parental line. A number of mechanisms have been shown to influence GR-DNA binding. For example, the GR is subject to posttranslational modification which can alter the trans-activating efficacy, including sumoylation and phosphorylation [39,40]. Furthermore, GC-resistant ALL primary samples have been associated with a decreased expression of subunits of the SWI/SNF chromatinremodeling complex required for GR transcriptional activity [41]. This is the first study to address the role of the transcription factor, NFB, in glucocorticoid resistance in a leukemic cell line model and suggests that despite differential induction of NFB modulators, NFB does not underlie the GC-resistant phenotype. Further investigation of the impact of the GC-sensitive associated proteins on other survival pathways, such as the RAS-RAF-MEK-ERK pathway, is warranted. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements The authors gratefully acknowledge Leukaemia Research for funding this study and thank Reinhard Kofler for providing the cell lines. Contributions. JI, CR and AH initially conceived the study and LN performed all experiments, analysed data and contributed to the study design. LN and JI drafted the article and all authors critically appraised and approved the final version. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.leukres.2009.12.014. References [1] Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Haqq C, Yamamoto KR. Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci USA 2004;101:15603–8. [2] Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA 1994;91:752–6. [3] Herr I, Gassler N, Friess H, Buchler MW. Regulation of differential pro- and anti-apoptotic signaling by glucocorticoids. Apoptosis 2007;12:271–91. [4] Riehm H, Reiter A, Schrappe M, Berthold F, Dopfer R, Gerein V, et al. Corticosteroid-dependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83). Klin Padiatr 1987;199:151–60.
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