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

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

C Ploner et al. Leukemia. 2008 Feb.

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.

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Figures

Figure 1
Figure 1

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. 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.

Figure 2
Figure 2

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

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.

Figure 4
Figure 4

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.

Figure 5
Figure 5

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.

Figure 6
Figure 6

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.

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