1Department of Drug Activity, New Drug Development Center, Medical Innovation Foundation, Osong, Daejeon, Korea.
2Department of Internal Medicine, School of Medicine, Chungnam National University, Daejeon, Korea.
The C-X-C chemokine receptor 7 (CXCR7) has been shown to be a decoy receptor for CXCR4 in certain cell types. We investigated the expression status and functional roles of CXCR7 in acute myeloid leukemia (AML) cells
All AML cell lines examined in this study (U937, K562, KG1a, HL-60, and MO7e) and primary CD34+ cells obtained from patients with AML expressed
CXCR7 is involved in the regulation of autocrine CXCL12 in AML cells.
Stromal cell-derived factor 1 (SDF-1), also known as C-X-C motif ligand 12 (CXCL12), is a chemokine that is constitutively expressed and produced by bone marrow stromal cells (BMSCs). It induces the migration and homing of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) by signaling
CXCR4 was the only known receptor for CXCL12 until the orphan receptor RDC1 (later renamed CXCR7) was discovered as an additional receptor for this chemokine . Thereafter, CXCR7-deficient mice revealed cardiovascular defects and early postnatal lethality, but normal hematopoiesis . Despite the controversial roles of CXCR7, especially with respect to CXCR4, it has been shown that CXCR7 is expressed in many cancer cell types and is involved in the development and progression of various cancers . Additionally, overexpression of CXCR7 in cancer cells was shown to indicate poor prognosis in many types of cancers . Based on these observations, it has been proposed that CXCR7 may be a therapeutic target in some cancers.
Whereas CXCR7 protein is expressed by primitive red blood cells (RBCs) during murine embryonic development, in adult mammals, this protein is not expressed by normal peripheral blood (PB) cells . A recent study has shown that CXCR7 is expressed at very low levels on normal CD34+ human HPCs and does not play a direct role in their proliferation or migration; however, it is involved in the trafficking/ adhesion of human leukemic cells .
The roles of CXCR7 in the survival and growth of AML cells, however, are not fully understood. Thus, the aim of the present study was to determine the role(s) of CXCR7 expression in the survival and proliferation of AML cells. Using siRNA technology,
BM samples were obtained, with informed consent, from 5 patients with AML at the time of diagnosis. CD34+ cells were purified from PB using the MACS system (Miltenyi Biotec, Auburn, CA, USA). Only cell preparations that contained >95% CD34+, as assessed by flow cytometry, were used in the experiments.
The human AML lines U937, HL-60, K562, and KG1a were purchased from the American Type Culture Collection (Manassas, VA, USA). The U937 cells were cultured in RPMI-1640 medium (Gibco-BRL Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL Life Technologies, Grand Island, NY, USA). The K562, KG1a, and HL-60 cells were grown in Iscove's modified Dulbecco's medium (IMDM; Gibco-BRL Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS. The MO7e cells were grown in IMDM supplemented with 10% FBS and 10 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA). For hypoxic exposure, cells were incubated with 5% CO2 and 1% O2 (v/v), balanced with N2 gas, at 37℃ for the time-periods indicated. The recombinant human CXCL12 and cytokines examined in this study were purchased from R& Systems. The following cytokines were used: interleukin-6 (IL-6), IL-3, granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ).
The cells were incubated with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated monoclonal antibodies at 4℃ for 30 min and analyzed using a Coulter Elite flow cytometer (Coulter Electronics Ltd., Hialeah, FL, USA) or a FACSCanto II flow cytometer (BD Pharmingen, San Diego, CA, USA). The monoclonal antibodies used were FITC-conjugated anti-CXCR4, PE-conjugated anti-CXCR4 (clone 12G5; BD Pharmingen, San Diego, CA, USA), and APC-conjugated anti-CXCR7/RDC-1 (clone 11G8; R&D Systems, Minneapolis, MN, USA). To detect cytoplasmic CXCR4 or CXCR7 expression, the cells were permeabilized with a saponin-based reagent (BD Pharmingen, San Diego, CA, USA) and labeled. To detect apoptosis, cells were stained with FITC-conjugated Annexin V and analyzed by flow cytometry.
Cells grown on coverslips (Paul Marienfeld Gmbh & Co. KG, Lauda-Koenigshofen, Germany) were washed with cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (Sigma-Aldrich Co., Saint Louis, MO, USA) for 15 min at 37℃, and washed 3 times with PBS. The cells were then incubated with a murine monoclonal anti-CXCR4 antibody (1:2,000; clone 12G5; R&D Systems, Minneapolis, MN, USA) and a murine monoclonal anti-CXCR7/RDC-1 antibody (1:2,000; clone 11G8; R&D Systems, Minneapolis, MN, USA) for 90 min at 37℃, washed 3 times with PBS, and incubated with FITC- or PE-conjugated anti-mouse IgG (1:4,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 37℃ for 60 min. The cells were washed 3 times with PBS, fixed, mounted on glass slides with PBS, and observed under a laser scanning confocal microscope (Olympus Corp., Lake Success, NY, USA).
For the transmigration experiments, cells (2×105 cells/well) were loaded into the upper chamber of a 24-well Transwell plate containing a 5 µm microporous membrane (Corning-Costar, Tewksbury, MA, USA), and the cells were allowed to migrate into the lower chamber containing 200 ng/mL CXCL12 for 4 hr. The migrated cells were counted, and the percentage of migrated cells or fold-increase in the number of migrated cells relative to that of the control (i.e., the migration index) was calculated.
Cell proliferation was measured using a colorimetric assay kit (CCK-8 assay kit; Dojindo Laboratories, Tokyo, Japan) according to the manufacturer's instructions. Briefly, 5×103 cells were incubated in 96-well plates in serum-free X-VIVO medium (BioWhittaker, Walkersville, MA, USA). After incubation, 10 µL CCK-8 solution, provided by the manufacturer, was added to each well. The optical density (OD) was measured 3 hr later using a spectrophotometer (Molecular Devices Co., Sunnyvale, CA, USA). The proliferation index represents the fold increase in the OD of the experimental cell solution from that of the control. The relative proliferation index represents the fold increase in the OD of the experimental cell solution from that of the control at the beginning of the incubation.
Total RNA was prepared from cells using TRIzol reagent (Gibco-BRL Life Technologies, Grand Island, NY, USA), according to the manufacturer's instructions. After purification, 1 µg RNA was reverse-transcribed using SuperScript reverse transcriptase (Gibco-BRL Life Technologies, Grand Island, NY, USA) and the universal primer oligo (dT)15 (Promega, Madison, WI, USA). In each reaction, 1 µL of cDNA was added to 24 µL PCR buffer (Gibco-BRL Life Technologies, Grand Island, NY, USA) supplemented with 2 mM MgCl2, 0.2 µM of each primer, and 1-U Koma Taq polymerase (Koma International, Seoul, Korea). Using a GeneAmp PCR system (Perkin Elmer, Norwalk, CT, USA), 30 cycles of 1 min at 94℃, 45 sec at 55-65℃, and 1 min at 72℃ were performed. The following primers were used: human
The quantification of human
Western blotting was used to detect CXCL12. Cells were starved in serum-free medium for 12 hr and then collected by centrifugation, washed in PBS, and lysed using sodium dodecyl sulfate (SDS) sample buffer (187.5 mM Tris-HCl, pH 6.8, 6% (w/v) SDS, 100% glycerol, 150 mM dithiothreitol, and 0.03% (w/v) bromophenol blue). Equal amounts of protein from each sample were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Amersham Life Science, Arlington Heights, IL, USA). The membranes were blocked for 1 hr in Tris-buffered saline (TBS) containing 5% (w/v) milk and 0.1% Tween 20 and then incubated with a primary mouse monoclonal antibody (Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4℃. The blots were washed with TBS containing Tween 20, incubated with the secondary antibody for 2 hr, and developed using West-Zol Plus (iNtRON Biotechnology, Seoul, Korea). Anti-mouse CXCL12 polyclonal antibody (Thermo Scientific, Barrington, IL, USA) was used.
Cells were seeded in 12-well plates (3×105 cells/well), incubated for 5-10 min at room temperature, and then transfected with 5-25 nM
Results are expressed as the mean±standard deviation (SD) of at least 3 experiments. Data were analyzed using Student's
All AML cell lines examined in this study (U937, K562, KG1a, HL-60, and MO7e) expressed both
CXCL12 induced the internalization of cell surface CXCR7 in the U937 cells (Fig. 2). Subsequently, we examined whether certain cytokines affected the expression of CXCR7 in AML cells. The AML cell lines were incubated with cytokines at various concentrations for up to 72 hr and then CXCR7 expression on the cell surface was examined using flow cytometry. The hematopoietic growth factors (IL-1β, IL-3, IL-6, G-CSF, GM-CSF, and SCF) and proinflammatory cytokines (IFN-γ, TGF-β, and TNF-α) examined did not alter the cell surface CXCR7 expression on the U937 cells. Similar results were obtained in the K562, KG1a, HL-60, and MO7e cells (data not shown). Exposure of the U937 cells to hypoxia (5% CO2 and 1% O2, balanced with N2 gas) at 37℃ for up to 12 hr did not alter the CXCR7 expression in these cells (Fig. 3). Similar results were obtained in the K562, KG1a, HL-60, and MO7e cells (data not shown).
To examine the role of CXCR7 in the migration of AML cells, we knocked down
To examine the roles of CXCR4 and CXCR7 in the survival and proliferation of AML cells, we knocked down
We examined whether knockdown of
All AML cell lines and primary AML cells examined in this study expressed CXCR7, both in the cytoplasm and on the cell surface at various levels, indicating that most AML cells express CXCR7. Hypoxia and ischemia are known to upregulate CXCR4 expression in most tissues as a tissue repairing mechanism . In contrast, the effects of ischemia on CXCR7 expression do not seem to be uniform. For example, hypoxic injury led to enhanced CXCR7 expression in neuronal cells  and mesenchymal stem cells ; however, it did not alter the CXCR7 expression in colon cancer cells . In the present study, hypoxia did not affect the CXCR7 expression in AML cells, confirming that the effects of hypoxia on CXCR7 expression differ among cell types. Various cytokines, including hematopoietic growth factors and proinflammatory cytokines, did not alter the CXCR7 expression in the AML cells. These results suggest that the regulatory mechanisms for CXCR7 are unique and different from those of CXCR4, and thus CXCR7 in AML cells has different roles from those of CXCR4.
In the present study, we found that CXCR7 was not involved in the CXCL12-mediated chemotaxis of AML cells, which has been shown in many other cell types. CXCR7 has previously been shown to enhance survival and growth in many cancer cell types. For example,
We have previously shown that AML cells produced CXCL12 and that knockdown of
Regarding the interaction between CXCR4 and CXCR7, several modes of CXCL12 signaling have been proposed. In certain cells, all effects initiated by CXCL12 are dedicated to signaling
In summary, we have shown that most AML cells express not only CXCR4 but also CXCR7 and that CXCR7 plays a role in the regulation of autocrine CXCL12 in AML cells.
Acute myeloid leukemia (AML) cells express and produce C-X-C chemokine receptor 7 (CXCR7). (
Abbreviations: CXCL12, C-X-C motif ligand 12; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
C-X-C motif ligand 12 (CXCL12) induces internalization of cell surface C-X-C chemokine receptor 7 (CXCR7) in acute myeloid leukemia (AML) cells. Flow cytometric analysis for cell surface CXCR7 in U937 cells before and after a 2-hr incubation with CXCL12 (200 ng/mL).
Cytokines and hypoxia do not alter C-X-C chemokine receptor 7 (CXCR7) expression in acute myeloid leukemia (AML) cells. (
Abbreviations: IL-6, interleukin 6; GM-SCF, granulocyte-macrophage colony-stimulating factor; TGF-β, tumor growth factor-β; INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor; TPO, thrombopoietin; Con, control.
C-X-C chemokine receptor 7 (CXCR7) does not affect migration, serum-deprivation-induced apoptosis, or spontaneous proliferation of acute myeloid leukemia (AML) cells. Using small interfering RNA (siRNA) technology,
Knock-down of C-X-C chemokine receptor 7 (CXCR7) upregulates C-X-C motif ligand 12 (CXCL12) expression in acute myeloid leukemia (AML) cells. Using small interfering RNA (siRNA) technology,