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. Author manuscript; available in PMC: 2016 Jul 19.
Published in final edited form as: Leukemia. 2007 Nov 29;22(2):370–377. doi: 10.1038/sj.leu.2405039

The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia

C Ploner 1, J Rainer 2, H Niederegger 2, M Eduardoff 2, A Villunger 3, S Geley 1, R Kofler 1,2
PMCID: PMC4950962  EMSID: EMS31329  PMID: 18046449

Abstract

Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival ‘rheostat’ is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.

Keywords: BCL2 rheostat, glucocorticoid-induced apoptosis, acute lymphoblastic leukemia

Introduction

Glucocorticoids (GCs) induce apoptosis in certain lymphoid cells and play an important role in the treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies.1 This effect is mediated by the GC receptor (GR), a ligand-activated transcription factor of the nuclear receptor superfamily that resides in the cytoplasm and, upon ligand binding, translocates into the nucleus, where it modulates gene expression via binding to specific DNA response elements or by protein–protein interactions with other transcription factors.2 A large number of genes have been identified that are regulated by GC in experimental systems of GC-induced apoptosis3 and related clinical samples,4,5 but the genes responsible for cell death induction remain controversial (for recent reviews see Schmidt et al.,3 Distelhorst,6 Schaaf and Cidlowski7 and Planey and Litwack8).

GC might induce cell death by directly regulating genes controlling cell survival and apoptosis, or via (de)regulating genes or gene networks leading to cellular distress which, in turn, constitutes an apoptotic stimulus. In both scenarios, members of the large family of pro- and anti-apoptotic BCL2 proteins,9,10 referred to as the ‘BCL2 rheostat’, might be involved either as direct GR targets or as sensors for potentially harmful GC effects.11 In addition, the status of the BCL2 rheostat, regardless of whether altered during GC exposure or not, might define sensitivity to, and kinetics of, GC-induced cell death. The latter issue was addressed in great detail in mice showing that GC-induced thymocyte apoptosis was impaired by transgenic expression of anti-apoptotic or knockout of some pro-apoptotic BCL2 family members (reviewed in Distelhorst,6 Strasser,9 Ranger et al.10 and Labi et al.12). In human ALL, only a few of these genes have been functionally tested in this respect. For instance, overexpression of BCL213 and knock-down of the BCL2 homology domain 3 (BH3)-only molecule BCL2L11/Bim14,15 interfered with, while overexpression of pro-apoptotic BAX16 and knock-down of anti-apoptotic MCL117 sensitized for, GC-induced apoptosis in ALL cell lines (following a recommendation by the HUGO Gene Nomenclature Committee, all official gene symbols are represented by uppercase letters to distinguish them from their alternatives, for example, BCL2L11 = Bim). To what extent, if any, expression of BCL2 family proteins predicts GC sensitivity in patients with ALL is controversial (discussed in Schmidt3), although MCL1 has recently been suggested as major GC resistance gene that specifically protects ALL cells from GC-induced, but not chemotherapy-induced, apoptosis.17 Taken together, the current evidence, strong in mice but less convincing in human systems, suggests that the status of the BCL2 rheostat influences GC sensitivity.

Concerning the question of whether components of the BCL2 rheostat might be regulated by GC, several BCL2 family members responded to GC in numerous systems of GC-induced apoptosis, most notably Bim, which was induced in mouse thymocytes,4,18 several leukemia cell lines,3,6,18 primary chronic lymphocytic leukemia cells19 and some patients with ALL.4 Other reported regulations include BMF and Puma mRNA induction in mouse thymocytes4,20 or BCL2 and Bcl-XL protein repression in children with ALL.21 However, in a recent study with primary ALL cells from children treated with GC ex vivo, neither Bim nor any other BCL2 family member was significantly regulated.5 The most critical question, that is, to what extent the BCL2 rheostat responds to GC treatment in patients in vivo, has not been thoroughly addressed.

In this study, we performed a systematic analysis of mRNA expression and GC regulation of all BCL2 family members during the early phase of systemic GC mono-therapy in children with ALL and complemented it with extended functional analyses in CCRF-CEM cells, a widely used model for GC-induced apoptosis in childhood ALL.

Materials and methods

The details of this section are available online as Supplementary Information.

Results

The BCL2 rheostat in the initial phase of GC treatment

To assess expression and possible regulation of the BCL2 rheostat in response to initial GC therapy in patients with ALL, we utilized our recently established expression profiles (Affymetrix, U133 plus 2.0) of lymphoblasts from 13 ALL children prior to, and 6–8 and 24 h after, initiation of GC treatment.4 First, all arrays were re-normalized using GCRMA (robust multi-array average with background adjustment using sequence information), which has been shown to be superior compared to the previously used RMA (robust multiarray analysis) normalization procedure. Second, we checked all probe sets (collection of eleven 25-mer oligonucleotides on the array that recognize a given transcript) assigned to the 21 genes of the BCL2 family9,10 for correct annotation and position along the transcripts and found proper probe sets (that is, probe sets that mapped within ~600 bp of the 3' end of the respective transcript) for all the 21 genes and almost all of their 42 RefSeq transcripts. The exceptions were one variant each of BCL2L14/Bcl-G and BCL2L11/Bim and three variants of the C1orf178/Bfk gene (Supplementary Table S1). Prior to treatment, MCL1 and BNIP3L were strongly expressed in all 13 childhood ALL samples, whereas BCL2L10/Boo/Diva, BOK and Bcl-G were not detectable in any of them (Figure 1a). The mRNA expression pattern seen in the children was largely maintained in the CCRF-CEM and PreB697 ALL in vitro models. The mean regulations (M-values, log2-fold changes) of the rheostat components in the childhood ALL samples along with their significance (pBH, P-values adjusted for multiple hypothesis testing according to Benjamini and Hochberg) are depicted in the ‘volcano plots’ in Figure 1b, which visualize regulations on the x axis and significance of these regulations on the y axis. At the early time point (6–8 h after treatment initiation), the BH3-only molecule BMF was the only significantly regulated BCL2 family member (pBH = 0.004, mean M = 0.8), but four additional genes came close to the significance cutoff pBH = 0.05, including the pro-survival gene BCL2A1/A1 (pBH = 0.057) and the pro-apoptotic HRK (pBH = 0.057) and PMAIP1/Noxa (pBH = 0.055) genes. Interestingly, A1 (mean M = 0.7) was induced and HRK (mean M = −0.3) and Noxa (mean M = −1.0) were repressed, presumably reflecting GC-mediated pro-survival signals. The fourth transcript, encoding the pro-apoptotic BAK1 molecule (pBH = 0.055), was induced, but the extent of regulation was very low (mean M = 0.2). After 24 h, the significance and mean M-values of A1, HRK and BAK1 decreased and another pro-apoptotic transcript was repressed (BAX, mean M = −0.5, pBH = 0.038). BMF regulation, in contrast, became more pronounced (mean M = 1.2, pBH = 0.003) and another BH3-only molecule, BCL2L11/Bim, reached significance levels (pBH = 0.038, mean M = 1.1).

Figure 1.

Figure 1

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Expression and regulation of BCL2 family members in ALL cells. (a) Expression (U133 plus 2.0-derived E-values, log 2 scale) of BCL2 family members in untreated malignant lymphoblasts from 13 children and 2 in vitro models (CEM-C7H2, PreB697). An intensity scale is indicated below the graph. E-values and probe sets for this graph are depicted in Supplementary Table S3. (b) mRNA regulations of BCL2 family members in peripheral blasts from 13 ALL children at 6–8 and 24 h of systemic GC monotherapy.4 Extent of regulation (mean M) was plotted against significance (Benjamini–Hochberg adjusted P-values, expressed as negative logarithm to the power of 10). The dotted lines indicate significance of pBH= 0.05 and regulation of M =±1. Genes with pBH values p ⩽0.05 are indicated. M-values and probe sets for the two ‘volcano plots’ are depicted in Supplementary Table S4. ALL, acute lymphoblastic leukaemia.

Since statistical procedures like the one above may obscure events in subgroups of patients, we also analyzed the response of individual children (Supplementary Table S5). Using >2-fold regulated in four or more individuals as an arbitrary cutoff, six BCL2 family members fulfilled this criterion, namely BCL2, A1, HRK, Noxa, Bim and BMF. BMF and Bim showed the most consistent regulations, that is, 11/13 patients induced BMF and/or Bim (M⩾1) and two of the remaining three showed borderline regulation (M = 0.8 and 0.9, after 24 h). One more BH3-containing transcript from the BCL2L11/Bim locus (called ‘Bam’) was induced in several patients and both cell lines (Supplementary Table S5). However, Bam is not a RefSeq mRNA and it is not known whether it is translated into a protein (see Discussion for more details). Thus, the clinical response of the BCL2 rheostat to GC treatment was dominated by induction of the pro-apoptotic BH3-only members BMF and Bim, but was otherwise heterogeneous.

Functional significance of Bim and BMF induction

To determine whether Bim and/or BMF induction is required for GC-induced cell death, we conditionally knocked down the expression of each of the two molecules in CCRF-CEM cells. As shown in Supplementary Table S5 on the mRNA level and in Figure 2 on the protein level, these cells induced both Bim and BMF after exposure to GC but prior to occurrence of cell death (Supplementary Figure S1). For functional analyses, we first generated a CCRF-CEM derivative expressing the tetracycline-responsive transrepressor tetR-KRAB (CEM-C7H2-2B10), transduced it with lentiviral constructs expressing small hairpin RNAs (shRNAs) directed against Bim or BMF in a tetracycline-dependent manner and generated stable derivatives by limiting dilution cloning. For each knock-down, two clones were analyzed (termed 2B10/Bim-shRNA#1, 2B10/Bim-shRNA#2, 2B10/BMF-shRNA#1 and 2B10/BMF-shRNA#39). These clones had maintained a normal GC response, as suggested by intact GR auto-induction, induction of FKBP51 and repression of cyclin D3 (Supplementary Figure S2), three known GC response genes in lymphoid cells. After induction of shRNA expression with doxycycline, both Bim-shRNA clones showed reduced basal and GC-induced levels of the target protein, with 2B10/Bim-shRNA#1 showing somewhat better knock-down than 2B10/Bim-shRNA#2 (Figure 3a, left panel). Bim knock-down was associated with a corresponding reduction in GC-induced apoptosis at 48 h (Figure 3b, left panel). The protective effect vanished after another 24 h, which may be explained by the inability of the shRNA to maintain Bim repression in the continuous presence of GC (data not shown) and/or by other pro-apoptotic regulatory events, such as BMF induction (see below). Knock-down of BMF almost completely prevented its induction by GC (Figure 3a, right panel); however, the protection from GC-induced cell death was less complete than that observed with Bim knock-down (Figure 3b, right panel). Taken together, the data suggested that induction of both Bim and BMF contributes to GC-induced apoptosis but that, in this model, Bim responds earlier and contributes more significantly to death induction than BMF.

Figure 2.

Figure 2

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Expression and GC regulation of BCL2 proteins in CEM-C7H2 T-ALL cells. CCRF-CEM-C7H2 cells were treated with 100 nM dexamethasone for 36 h (a and b) or for the indicated times (c) and analyzed by immunoblotting using antibodies specific for the indicated pro- (a) and anti- (b) apoptotic BCL2 proteins. The asterisk marks a recently identified new BMF isoform (Villunger et al., in preparation).

Figure 3.

Figure 3

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Effect of conditional Bim or BMF knock-down on GC-induced apoptosis. (a) CEM-C7H2-2B10 subclones conditionally expressing shRNA small hairpin RNA (shRNA) targeting Bim or BMF were cultured for 3 days in the presence or absence of 500 ng ml−1 doxycycline (Dox) and subsequently exposed to 100 nM dexamethasone (Dex) or 0.1% ethanol as carrier control for up to 72 h. Bim and BMF expression was monitored after 24 h (Bim) or 30 h (BMF) exposure to dexamethasone by immunoblotting using α-tubulin (α-Tub) as loading control. (b) Extent of GC-induced apoptosis was assessed by FACS analysis of propidium iodide-stained nuclei at the times indicated. Bars represent the means±s.d. of at least four independent experiments.

To assess whether induction of Bim or BMF alone is sufficient to induce cell death, we generated stable derivatives of CEM-C7H2-2C8 cells (which constitutively express the tetracycline-regulated transactivator protein rtTA)22 by lentiviral transduction with constructs enabling tetracycline-induced expression of transgenic BimEL (NM_138621) and BMF-1 (NM_001003940), the two major GC-regulated isoforms in this system (Figure 4). In these cell lines, the level of transgene expression could be controlled at will by varying the amount of doxycycline (Figure 4a). Using this system, overexpression of both BimEL and BMF-1 led to significant cell death in a dose–dependent manner (Figure 4b), proving the ability of these molecules to induce apoptosis in the model cell line and, by extrapolation, probably in ALL cells from patients as well.

Figure 4.

Figure 4

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Effect of conditional BimEL or BMF-1 transgene expression on cell survival. CEM-C7H2-2C8 derivatives conditionally expressing transgenic BimEL (2C8/BimEL#17) or BMF-1 (2C8/BMF1#8) were cultured in the presence of the indicated doxycycline concentrations and analyzed by immunoblotting after 3 h (a) and by flow cytometry of propidium iodide-stained nuclei to determine the degree of apoptosis after 24 h (b). FACS data shown represent the arithmetic means±s.d. of three independent experiments.

Bim binds to and stabilizes MCL1

The marked induction of the MCL1 protein that followed the kinetics of the Bim protein induction (Figure 2c) raised the questions of whether MCL1 induction was related to that of Bim, and why MCL1 induction did not prevent apoptosis. In response to the former, GC induction of the MCL1 protein was reduced when the parallel induction of Bim was prevented by shRNA-mediated knock-down (Figure 5a). Moreover, tetracycline-induced Bim expression resulted in increased MCL1 protein in the absence of GC (Figure 5b). Taken together, these data indicate that the increase of MCL1 protein observed after GC exposure was, at least in part, a consequence of GC-mediated induction of Bim, presumably via MCL1 stabilization, as suggested by monitoring its expression in cycloheximide-treated cells in the absence or presence of GC (Figure 5c). The latter conclusion was further supported by the recent observation that transfected human BimS caused endogenous MCL-1 protein stabilization in mouse embryonic fibroblasts.23 The failure of increasing MCL1 protein to block apoptosis can be explained by the observation that significantly more Bim co-immunoprecipitated with MCL1 after GC treatment (Figure 5d). Thus, unlike Noxa, which binds to MCL1 and facilitates its degradation,24 Bim appears to bind to and inactivate MCL1 without degradation.

Figure 5.

Figure 5

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MCL1 upregulation during GC treatment. (a) 2B10/Bim-shRNA#1 cells pre-cultured in the presence or absence of 500 ng ml−1 doxycycline for 72 h were treated with 100 nM dexamethasone or 0.1% ethanol for another 24 h and analyzed by immunoblotting using antibodies against Bim, MCL1 and α-tubulin as loading control. (b) C7H2-2C8 (left panel) and its derivative 2C8/BimEL#9 (right panel) were treated for the times indicated with 100 nM dexamethasone or 12.5 ng ml−1 doxycycline, respectively, and analyzed by immunoblotting using antibodies against Bim, MCL1 and α-tubulin. (c) CCRF-CEM-C7H2 cells were pre-treated with 100 nM dexamethasone or 0.1% ethanol as control for 24 h, then cultured in the presence or absence of 10 µM cycloheximide for the indicated times and subjected to immunoblotting with antibodies against MCL1 or α-tubulin as loading control. (d) CCRF-CEM-C7H2 cells were treated with 100 nM dexamethasone for 24 h and cell lysates were immunoprecipitated with anti-MCL1 antibodies (IP: MCL1) or normal rabbit serum as control (IP: control). Aliquots of immunoprecipitates were subjected to immunoblotting with anti-MCL1 (IB: MCL1) or anti-Bim (IB: BimEL) antibodies.

Effect of the BCL2 rheostat on GC sensitivity

To determine how the status of the BCL2 rheostat prior to treatment affects sensitivity and kinetics of GC-induced apoptosis, we genetically manipulated the expression levels of MCL1, Bcl-XL and BCL2, the three anti-apoptotic BCL2 family members expressed in this ALL model (Figures 1a and 2). First, we generated clonal CCRF-CEM derivatives with conditional knock-down of either of the three molecules. Doxycycline exposure for 72–96 h led to a significant reduction in the expression of MCL1, Bcl-XL and BCL2 (Figure 6a). This reduction alone had no detectable effect on cell cycle progression and viability during the first 72 h, but in all three instances, the cells became more sensitive to GC-induced apoptosis and the kinetics of the response was accelerated (Figure 6b). The effect was most pronounced with MCL1 knock-down where cells died 24 h earlier and responded to as little as 10 nM dexamethasone. Bcl-XL knock-down showed an intermediary response and that of BCL2 the weakest.

Figure 6.

Figure 6

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Effect of knock-down and overexpression of anti-apoptotic BCL2 proteins on GC-induced apoptosis. (a) CEM-C7H2-2B10 (expressing tetR-KRAB) derivatives conditionally expressing small interfering RNAs (shRNAs) targeting Bcl-XL (C7H2-2B10/BclX-shRNA-#8 and #13), BCL2 (C7H2-2B10/BCL2-shRNA-#3 and #4) or MCL1 (C7H2-2B10/MCL-shRNA-#6 and #11) were cultured with 500 ng ml−1 doxycycline for the indicated time and analyzed by western blotting with antibodies specific for the indicated BCL2 family members. Shown are data from one subclone each. (b) The same cell lines were pre-cultured for 72 h in the absence or presence of 500 ng ml−1 doxycycline (Dox), the cultures continued for another 24 h with and without dexamethasone (Dex, 100, 50 or 10 nM) and apoptosis determined by flow cytometry of propidium iodide-stained nuclei. The data shown represent the arithmetic means±s.d. of experiments performed in triplicate with both clones for each gene. (c) CEM-C7H2-2C8 (expressing rtTA) and its derivatives with conditional expression of transgenic MCL1 (2C8/MCL1#3, #11, #20) were cultured in the absence or presence of 100 nM dexamethasone (Dex, 2C8 only) or 100 ng ml−1 doxycycline (Dox, all others) for 24 h and MCL1 expression monitored by immunoblotting (left panel). Similarly, Bcl-XL expression was determined in CEM-C7H2 cultured in the absence or presence of 100 nM dexamethasone (Dex, C7H2 only) and in its untreated derivatives with constitutive Bcl-XL expression (C7H2/BclXL-2F1, 2F10 and 2G9, right panel). (d) MCL1 overexpressing (+ Dox) and control (−Dox) cells (left panel) and Bcl-XL-overexpressing and parental CEM-C7H2 cells (right panel) were treated with 100 nM dexamethasone for the indicated time and analyzed by flow cytometry of propidium iodide-stained nuclei. Data show means of three independent experiments±s.d.

In the second approach, we investigated whether increased expression of these molecules protected against GC-induced apoptosis. We had previously reported that doxycycline-controlled BCL2 expression delayed GC-induced cell death in CEM-C7H2 cells by about 24 h.13 Figures 6c and d show a similar effect for both Bcl-XL and MCL1, with the degree of protection roughly correlating with the amount of transgene expression. Thus, among the MCL1-overexpressing cell lines, 2C8/MCL1#13 cells showed the least transgene induction upon doxycycline exposure and the weakest protection. 2C8/MCL1#11 and #20 expressed more MCL1 and were better protected. The three Bcl-XL cell lines expressed their transgene at very high levels, which entailed an almost 48 h delay in GC-induced apoptosis. Taken together, these data indicated that expression levels of all the three anti-apoptotic BCL2 family members prior to GC treatment influence sensitivity and kinetics of the GC-induced cell death response.

Discussion

The present study delineated, for the first time, expression and regulation of the entire BCL2 rheostat in the early phase of GC therapy in ALL patients and compared it with the corresponding response in two well-defined ALL in vitro models. The clinical response differed between individual patients and encompassed both pro- and anti-apoptotic signaling. Nevertheless, the BH3-only genes BMF and/or Bim were induced in the vast majority of ALL children as well as an adult with ALL. In addition, another BH3-containing transcript from the BCL2L11 locus, termed Bam,25 was induced (M⩾1) in 10/14 patients (due to space limitations we discuss this transcript and the question of whether the regulated BCL2 genes are direct GC targets in the Supplementary Information).

Whether BMF and Bim induction is necessary and/or sufficient for cell death was addressed in the CCRF-CEM childhood ALL model. Despite the known limitations, an in vitro model had to be used because functional analyses like the ones performed in this study cannot currently be performed with primary cells because of technical difficulties and the fact that ALL blasts undergo rapid spontaneous apoptosis in vitro. It was not surprising that transgenic Bim caused apoptosis, since Bim is considered one of the most potent pro-apoptotic molecules in both current BCL2 rheostat models. In the ‘direct activator/derepressor model,’26,27 it acts as a direct activator of BAX, and in the ‘displacement model’,12,28,29 it is a potent neutralizer of all five BCL2-like pro-survival proteins. That BMF on its own, and at levels comparable to those seen after GC induction (Figures 2 and 3), sufficed for apoptosis induction was less predictable. In the first model, sole induction of BMF as a ‘de-repressor’ should not entail cell death; however, since a ‘direct activator’ (in our case Bim; Figure 2) is already present in the system, a ‘de-repressor’ might suffice for cell death induction.30 In the ‘displacement model’ BMF is considered a weak death agonist that neutralizes only BCL2, Bcl-X or Bcl-W, but not A1 or MCL1,31 the latter being well expressed in CCRF-CEM cells (Figures 1 and 2). However, since Bim and Noxa are expressed as well (Figure 2) and may neutralize MCL1, transgenic BMF might kill by freeing pro-apoptotic BAK out of its complex with Bcl-XL, as recently suggested.24 In any case, if the data of the CCRF-CEM model can be extended to the clinical situation, the induction of BMF and/or Bim would explain GC-induced cell death in the majority of patients on a mechanistic level. In this context, it is worth mentioning that Puma mRNA, which is induced by GC in mouse thymocytes in vivo and ex vivo4,20 and whose knockout impairs GC-induced thymocyte apoptosis,32,33 was not regulated in the investigated patients (Supplementary Table S5). Thus, the human Puma gene is not a transcriptional target of the GR, and if Puma contributes to GC-induced apoptosis in human ALL, it does so in a transcription-independent manner.

A question of considerable clinical relevance is whether expression of BCL2 family proteins predicts GC responsiveness in ALL patients. The experiments in Figure 6 considered together with similar data in the literature13–16 indicate that the status of the BCL2 rheostat prior to treatment affects the kinetics of, and sensitivity to, GC-induced apoptosis in experimental systems. In contrast, the literature concerning this issue in patients has been controversial,3 although a recent report combining bioinformatic analyses of expression profiles from ALL children (classified as GC-sensitive/resistant by ex vivo testing) with functional analyses in experimental systems identified MCL1 as the key anti-apoptotic BCL2 family protein responsible for GC resistance.17 However, direct proof that MCL1 expression, but not that of other pro-survival proteins, predicts and causes in vivo resistance to GC therapy is still lacking. Interestingly in this respect, a recent genome-wide gene expression comparison between precursor B-cell blasts at diagnosis and after 8-day systemic GC monotherapy showed differential expression of a single BCL2, family member, that is, BCL2 which was surprisingly reduced in day 8 blasts, which are considered to be GC resistant.34

In addition to pro-apoptotic signals (induction of Bim and BMF, repression of BCL2 and Bcl-XL), numerous patients and both cell lines showed anti-apoptotic regulations as well, most impressively a marked reduction of the BH3-only molecule Noxa, a specific antagonist of MCL1 implicated in growth factor withdrawal and nutrient shortage-induced cell death in the lymphoid lineage.35 This is reminiscent of the well-documented potential of GC to elicit cell death in some, but be protective in other, cell types.3,36 In the CCRF-CEM model, and perhaps in patients as well, anti-apoptotic and pro-apoptotic signaling may even occur within the same cell. The CCRF-CEM model may exemplify just one possible scenario: Noxa reduction may free MCL1, which in turn serves as a buffer for increasing Bim levels, thereby preventing cell death within the first 24 h. Thereafter, additional pro-apoptotic regulations (for example, further increase of Bim, induction of BMF and/or Puma, repression of BCL2 and/or Bcl-XL) might tip the balance. Thus, the cellular context and additional signals feeding into the BCL2 rheostat may ultimately determine which BCL2 family members participate in this antagonistic interplay, to what extent and in which direction they are being regulated and whether survival or cell death ensues.

In conclusion, our study suggests a model in which GCs affect, directly or indirectly, expression of the BCL2 rheostat, in particular that of the BH3-only molecules BMF, Bim and Noxa, thereby controlling the activity of anti-apoptotic BCL2 molecules, such as MCL1, and the killer proteins BAX and/or BAK. Depending on the cellular context (including levels and responsiveness of the GR,37 its phosphorylation status,38–40 expression of BCL2 genes prior to treatment and additional signals feeding into the rheostat), this effect may lead to different sensitivity to, and kinetics of, GC-induced cell death. With the advent of effective antagonists of anti-apoptotic BCL2 proteins, the emerging understanding of the BCL2 rheostat prior to and during therapy of ALL cells may become relevant for innovative therapy.30

Supplementary Material

Supplementary Data, Figures and Tables

Acknowledgements

We thank Drs A Helmberg, K Janjetovic and Z Trajanoski for stimulating discussions, Drs D Huang, A Strasser and G Nolan for reagents, S Jesacher and Mag S. Lobenwein for technical assistance, and MK Occhipinti-Bender for editing. This work was supported by grants from the Austrian Science Fund (SFB-F021, P18747, P18571), the Austrian Ministry for Education, Science and Culture (GENAU-Ch.I.L.D.) and the European community (LSHS-CT-2004-503438, TRANSFOG).

Footnotes

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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