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Clinical Trial
. 2013 Aug 9;288(32):22899-914.
doi: 10.1074/jbc.M113.449926. Epub 2013 Jun 24.

CXCR4 chemokine receptor signaling induces apoptosis in acute myeloid leukemia cells via regulation of the Bcl-2 family members Bcl-XL, Noxa, and Bak

Kimberly N Kremer  1 , Kevin L Peterson, Paula A Schneider, X Wei Meng, Haiming Dai, Allan D Hess, B Douglas Smith, Christie Rodriguez-Ramirez, Judith E Karp, Scott H Kaufmann, Karen E Hedin
Affiliations
Clinical Trial

CXCR4 chemokine receptor signaling induces apoptosis in acute myeloid leukemia cells via regulation of the Bcl-2 family members Bcl-XL, Noxa, and Bak

Kimberly N Kremer et al. J Biol Chem. .

Abstract

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.

Keywords: AML; Apoptosis; Bcl-2 Family Proteins; CXCL12; CXCR4; Cancer; Caspase; Chemokines; Leukemia; SDF-1.

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Figures

FIGURE 1.
FIGURE 1.
CXCR4 is expressed at variable levels on AML cells. A, bone marrow isolates were harvested from AML patients prior to chemotherapy. Patient samples were cultured for 16–18 h before levels of CXCR4 protein on the cell surface were analyzed via incubation of intact cells with anti-CXCR4 mAb followed by flow microfluorimetry. Results from nine different patient isolates are shown. B, to assay intracellular CXCR4 protein levels, cells from AML isolates were fixed, permeabilized, and then incubated with either a CXCR4 antibody or a control antibody prior to flow microfluorimetry. Results of a representative isolate are shown; n = 8. C, either endogenous, cell surface CXCR4 (left) or endogenous intracellular CXCR4 (middle) was assayed for the human AML cell line, KG1a; representative results are shown, n = 3. Right, cell surface CXCR4 levels of KG1a cells transfected with either a control plasmid (filled histogram) or a plasmid encoding the CXCR4-YFP fluorescent fusion protein (open histograms) are shown, either before (Unstim.) or after 20 min stimulation with SDF-1 (SDF-1) that induced CXCR4 internalization; representative results from one experiment are shown; n = 3.
FIGURE 2.
FIGURE 2.
SDF-1 signaling induces migration and ERK activation in the KG1a-CXCR4. KG1a cells were transfected with either a vector control plasmid or CXCR4-YFP as in Fig. 1C. A, transfected cells were assayed for migration in response to SDF-1. Each point denotes the mean percentage increase over basal migration in response to the indicated concentration of SDF-1 ± S.E. (error bars); n = 3. *, significantly different from KG1a cells transfected with the vector control plasmid; p < 0.05. B, transfected cells were assayed for active, phosphorylated ERK in response to treatment with SDF-1 for the indicated times in YFP-positive cells; representative flow cytometric plots from one experiment are shown. C, summary of the results of three independent experiments performed as in B; each point denotes the average geometric mean of active, phosphorylated ERK ± S.E. *, significantly different from KG1a cells transfected with the vector control plasmid and treated with SDF-1; p < 0.05.
FIGURE 3.
FIGURE 3.
SDF-1 induces KG1a cell apoptosis via CXCR4. KG1a cells transfected with either the vector control plasmid or CXCR4-YFP were cultured with SDF-1 for 16–18 h. A, cells were stained with APC-conjugated annexin V and assayed via flow microfluorimetry for apoptosis as indicated by annexin V binding to the cell surface. Gating as shown was used to compare apoptosis among cells expressing similar high levels of either CXCR4-YFP or YFP (vector control). B, summary of results from three independent experiments done as in A. Each bar denotes the percentage of cells positive for annexin V ± S.E. (error bars). *, significantly different from KG1a cells transfected with the vector control plasmid and treated with SDF-1; p < 0.05. C, apoptosis of SDF-1-treated cells was confirmed by confocal microscopic imaging of Hoechst 33258-stained nuclei. A representative image is shown; the arrow denotes nuclear fragmentation typically associated with apoptosis. D and E, cells were fixed and permeabilized and then stained with an antibody specific for either cleaved PARP or for cleaved (active) caspase-3 and analyzed by flow microfluorimetry. Gating as shown was used to analyze cells expressing similar levels of CXCR4-YFP. F, results of multiple independent experiments performed as in D and E. Each bar denotes the percentage of cells with each of the indicators of apoptosis ± S.E.; n = 3. *, significantly different from unstimulated cells; p < 0.05. G, cells were analyzed for the correlation between apoptosis as assayed by annexin V binding and expression of either CXCR4-YFP or YFP (vector control). Each point denotes the mean change in percentage of cells positive for annexin V in response to SDF-1 ± S.E.; n = 3. *, significantly different from KG1a cells transfected with the vector control plasmid and treated with SDF-1; p < 0.05. H, results of multiple experiments as in A, in which the concentration of SDF-1 was varied as indicated prior to an assay of annexin V binding. Each point denotes the mean percentage of cells positive for annexin V among cells expressing similar levels of CXCR4-YFP ± S.E.; n = 3. I, the indicated cells were treated with 30 μm AMD3100 (a CXCR4 antagonist) prior to stimulation with SDF-1. Following SDF-1 treatment, the cells were assayed for annexin V binding as in A. Each bar denotes the mean percentage of cells positive for annexin V ± S.E.; n = 3. *, significantly different from untreated KG1a cells transfected with CXCR4-YFP and stimulated with SDF-1; p < 0.05.
FIGURE 4.
FIGURE 4.
SDF-1/CXCR4 induces apoptosis in the U937 AML cell line and in a subset of clinical AML isolates. A, the AML cell line U937 was transfected with either a vector control plasmid (YFP) or CXCR4-YFP, cultured for 16–18 h, stained with an APC-conjugated CXCR4 antibody, and assayed for CXCR4 expression by flow microfluorimetry while gating on cells expressing similar levels of CXCR4-YFP or YFP. A representative flow cytometric plot is shown; n = 3. B, cells were transfected as in A and assayed for annexin V-positive cells as in Fig. 3A. *, significantly different from unstimulated CXCR4-transfected cells; p < 0.05. C, clinical AML isolates were cultured for 1–2 h and then plated onto a confluent layer of BMSC, cultured for 1 h, and then treated with 5 × 10−8 m SDF-1 for 16–18 h. Isolates were then stained with APC-conjugated annexin V and assayed for apoptosis by flow microfluorimetry as in Fig. 3A. Results for 10 different patients are shown. Error bars, S.E.
FIGURE 5.
FIGURE 5.
Hypoxia protects CXCR4-expressing AML cell lines from apoptosis. Either KG1a or U937 cells were transiently transfected with CXCR4-YFP to generate KG1a-CXCR4 or U937-CXCR4 cells, respectively. Cells were stimulated with SDF-1 for 16–18 h in ambient (normoxic) or 1% oxygen (hypoxic) conditions, as indicated. A and D, apoptosis was assayed by annexin V binding as in Fig. 3A. *, significantly different from unstimulated cells in normoxic conditions; p < 0.05. **, significantly different from SDF-1-treated cells in normoxic conditions; p < 0.05; n = 3. B and E, apoptosis was assayed by detection of cleaved PARP as in Fig. 3F. *, significantly different from unstimulated cells in normoxic conditions; p < 0.05. **, significantly different from SDF-1-treated cells in normoxic conditions; p < 0.05; n = 3. C and F, CXCR4 cell surface levels were assayed by flow microfluorimetry on the same unstimulated cells used for experiments as in A and D. *, significantly different from cell culture in normoxic conditions; p < 0.05; n = 3. G, untransfected KG1a cells were incubated in hypoxic or normoxic conditions for 2 h and then treated with 1 μm cytarabine for 18 h in normoxic or hypoxic conditions prior to analysis of annexin V binding. *, significantly different from cytarabine-treated cells in normoxic conditions; p < 0.05; n = 3. Error bars, S.E.
FIGURE 6.
FIGURE 6.
CXCR7, ERK activation, and Gi proteins do not mediate SDF-1-induced apoptosis in AML cells. A, CXCR7 cell surface levels were assayed by flow microfluorimetry on the indicated cell lines; representative results from one experiment are shown; n = 3. B, C, and F, KG1a-CXCR4 cells were pretreated with CCX771 (CXCR7 antagonist), CCX704 (the control), PD325901, CI-1040, or the vehicle for 1 h prior to the addition of SDF-1 or with PTX-B or PTX for 4 h prior to the addition of SDF-1. Cells were then cultured for 16–18 h and assayed for apoptosis via annexin V binding as in Fig. 3A; n = 3. D and E, KG1a-CXCR4 cells were treated with PD325901, CI-1040, or vehicle for 16–18 h, stimulated with SDF-1 for 2 min, and then assayed for ERK activation as in Fig. 2, B and C. *, significantly different from control-treated cells, p < 0.05; n = 3. G and H, KG1a-CXCR4 cells were cultured for 16 h, treated with PTX-B or PTX for 4 h, stimulated with SDF-1 for 2 min, and assayed for ERK activation as in Fig. 2, B and C. *, significantly different from SDF-1-stimulated control-treated cells, p < 0.05, n = 3. Error bars, S.E.
FIGURE 7.
FIGURE 7.
Requirement for the caspase-9/intrinsic death pathway but not the caspase-8/extrinsic pathway, for SDF-1/CXCR4-mediated apoptosis. A and C, KG1a or U937 cells were transiently transfected with CXCR4-YFP together with either a control plasmid (Vector) or a plasmid encoding either MC159 or DN-Casp-9-GFP. Cells were then assayed for SDF-1-dependent apoptosis via annexin V binding as in Fig. 3A. *, significantly different from SDF-1-treated control vector; p < 0.05; n = 3. B, Jurkat T cells were transiently transfected with a plasmid encoding either a control vector or the caspase-8 inhibitor, MC159, and then stimulated with agonistic anti-Fas antibody for 16–18 h and assayed for apoptosis via annexin V binding as in Fig. 3A. *, significantly different from the CH.11-treated control vector results; p < 0.05; n = 3. D, immunoblots of whole cell lysates from A and C, showing overexpression of DN-Casp-9-GFP as compared with endogenous Procasp-9. The same membrane was stripped and reblotted for actin as a control. E, mRNA from cells transfected as in A–C was assayed via RT-PCR for expression of the mRNA encoding the viral protein, MC159. GAPDH mRNA was assayed as a control; n = 3. Error bars, S.E.
FIGURE 8.
FIGURE 8.
Role of SDF-1-mediated Bak up-regulation in SDF-1/CXCR4-mediated apoptosis. A, KG1a cells were transfected with CXCR4-YFP and treated with SDF-1 for 16–18 h. The cells were sorted using a flow cytometer to obtain the top 12% of YFP-positive cells, and mRNA of the indicated Bcl-2 family members was assayed via quantitative RT-PCR. Each bar denotes the mean -fold increase of mRNA expression in response to SDF-1 treatment ± S.E. (error bars) as compared with unstimulated cells, following normalization of the results to the mRNA levels of GAPDH, for three independent experiments and sorts done on different days. *, significantly different from GAPDH control; p < 0.05. B, KG1a cells were transfected, treated, and sorted as in A, and Bak and ERK (control) protein expression levels were assayed in whole cell lysates via immunoblotting; n = 3. C and D, KG1a cells were transfected with either a GFP plasmid control vector or GFP-Bak, cultured for 24 h (together with the caspase inhibitor, Q-VD-OPh, where indicated), and analyzed for GFP expression levels via flow microfluorimetry (C) and annexin V positivity in these GFP-expressing cells (D). Representative experiments are shown. E, summary of results from three independent experiments performed as in D. Each bar denotes the percentage of annexin V-positive cells ± S.E. *, significantly different from control vector; p < 0.05. F and H, immunoblots showing depletion of Bak or Bim in the KG1a cell lines stably expressing Bak shRNA or Bim shRNA, respectively, as compared with the KG1a cell line expressing control shRNAs. The same immunoblots were stripped and reprobed with actin as a control; n = 3. G and I, KG1a cells stably expressing Bak shRNA, Bim shRNA, or the shRNA control vector were transiently transfected with CXCR4-YFP and then assayed for SDF-1-dependent apoptosis as in Fig. 3A. *, significantly different from SDF-1-treated control vector; p < 0.05; n = 5 and n = 2, respectively.
FIGURE 9.
FIGURE 9.
SDF-1-induced down-regulation of Bcl-XL and up-regulation of Noxa contribute to apoptosis of AML cells. A, KG1a cells were transfected, treated, and sorted as in Fig. 8A, and the protein expression levels of the indicated Bcl-2 family members were assayed via immunoblot. The same immunoblots were stripped and reprobed with actin as a control; n = 3. B, KG1a cells were transfected with CXCR4-YFP and either a control vector or S-peptide-tagged Bcl-XL (S-tagged-Bcl-XL) and assayed for Bcl-XL and actin (control) protein levels via immunoblot. C, KG1a cells were transfected as in B, cultured for 6 h, treated with SDF-1 for 18 h, and then assayed for annexin V positivity in YFP-expressing cells. Results from three independent experiments are shown. Each bar denotes the mean percentage of cells positive for annexin V ± S.E. (error bars). *, significantly different from control vector; p < 0.05. D–F, KG1a cells were transfected with a plasmid encoding either GFP or GFP-Noxa, cultured for 24 h (together with the caspase inhibitor, Q-VD-OPh, where indicated), and assayed for expression levels via Western blot (D), GFP expression via flow microfluorimetry (E), and annexin V positivity in GFP-expressing cells (F). Representative experiments are shown. G, summary of results from three independent experiments performed as in F. Each bar denotes the percentage of annexin V-positive cells ± S.E. *, significantly different from control vector; p < 0.05. H, KG1a cells were cotransfected with CXCR4-YFP and Noxa2A-GFP; cultured for 16 h in the presence or absence of SDF-1; and then treated with cycloheximide for the indicated time, fixed, and analyzed via flow microfluorimetry for Noxa2A-GFP expression in CXCR4-YFP-positive cells. Each point denotes the percentage of Noxa2A-GFP expression as compared with 0 h. *, significantly different from vehicle control; p < 0.05; n = 3. Inset, immunoblot of the indicated whole cell lysates confirming expression of Noxa2A-GFP compared with actin (control). I, immunoblots of whole cell lysates from KG1a transfected with CXCR4-YFP and an siRNA for Noxa or a control siRNA (ctrl), cultured for 24 h, and blotted for Noxa or actin (control). J, KG1a cells were transfected as in I, cultured for 6–7 h prior to the addition of SDF-1, and then cultured with or without SDF-1 for an additional 16 h and assayed for annexin V-positive cells as in Fig. 3A). Each bar denotes the percentage of annexin V-positive cells ± S.E. *, significantly different from SDF-1-stimulated cells treated with the control siRNA; p < 0.05.
FIGURE 10.
FIGURE 10.
Proposed model of SDF-1-induced apoptosis. SDF-1-induced CXCR4 activation induces Noxa stabilization, Bak up-regulation, and Bcl-XL down-regulation. Collectively, these lead to Bax/Bak activation followed by activation of caspase-9 and caspase-3. Consistent with this model, increased expression of Noxa or Bak is shown to induce apoptosis in these cells, and Bcl-XL expression, Bak shRNA, or Noxa siRNA is shown to inhibit SDF-1-induced apoptosis. Likewise, AMD3100 and DN-Casp-9 inhibit apoptosis, providing further support for the involvement of CXCR4 and the caspase 9-mediated intrinsic pathway in this process.
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