Abstract
Background: Signaling induced by binding of erythropoietin-producing hepatoma-amplified sequence (EPH) receptors to their cell-surface ephrin ligands is implicated in hematopoiesis and growth of various cancer cells. However, the roles of EPH–ephrin signaling in leukemia have not been elucidated. We investigated the effects of EPHB4 and ephrin B2 on the growth of leukemia cells. Materials and Methods: Seven human leukemia cell lines were used to examine the effects of recombinant ephrin B2 and EPHB4 on cell proliferation by colorimetric WST-1 assay and colony assays; on protein tyrosine phosphorylation; and on mRNA expression by reverse transcription-polymerase chain reaction and microarray analysis. Results: In an erythroid leukemia-derived cell line AA, exogenous ephrin B2 induced proliferation and colony formation; in addition, it up-regulated protein tyrosine phosphorylation and the expression of growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B and v-src avian sarcoma viral oncogene homolog. Conclusion: Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH–ephrin signaling may be involved in the pathology of leukemia.
Erythropoietin-producing hepatoma-amplified sequence receptors encoded by EPH genes are a family of receptor tyrosine kinases consisting of 14 members (EPHA1-8, A10, B1-4, and B6). EPH cell-surface ligands, ephrins, are encoded by the genes of the EFN family and consist of eight members (ephrins A1-5 and B1-3). The binding of an ephrin ligand to a respective EPH receptor activates bi-directional signaling, affecting cell proliferation and cell-fate determination in a number of key biological processes, including angiogenesis and hematopoiesis (1). In particular, EPHB4 and its corresponding ligand ephrin B2 are involved in erythropoiesis (2). Various types of cancer cells have been reported to express high levels of EPH receptors and ephrins (3). Although the expression of some EPH and EFN genes in leukemia cells has been detected (4-7), the functional significance of EPH–ephrin signaling in leukemia has not been fully-elucidated.
In our previous studies, we investigated the roles of stemness-related molecules such as NOTCH (8) and bone morphogenetic protein-4 (BMP4) (9) on the growth of leukemia cells. The aim of this study was to assess the expression of different EPH and EFN genes in leukemia cells and to evaluate the effects of ephrin B2 and EPHB4, closely associated with hematopoiesis (2, 10), on cell proliferation; the effect of an EPHB4 inhibitor was also examined. Furthermore, we investigated the relationship between EPH–ephrin and NOTCH signaling pathways because the cooperation of these pathways has been demonstrated in other developmental processes such as angiogenesis (11, 12).
Materials and Methods
Cells and reagents. Five acute myeloblastic leukemia (AML) cell lines (NB4, THP-1, TMD7, HEL, and AA), an acute T-lymphoblastic leukemia (T-ALL) cell line (Jurkat), and a B-lymphoma cell line (TMD8) were used in this study. NB4 cells (derived from acute promyelocytic leukemia) were kindly provided by Dr. M. Lanotte (Saint-Louis Hospital, Paris, France). THP-1 (monocytic leukemia) and HEL (erythroid leukemia) cells were obtained from the Health Science Research Resource Bank (Osaka, Japan) and the Japanese Cancer Research Resource Bank (Tokyo, Japan), respectively. AA cell line was established by Dr. A. Arai (Department of Hematology, Tokyo Medical and Dental University, Tokyo, Japan) from acute pure erythroid leukemia cells (13). Jurkat cells were purchased from the European Collection of Cell Cultures (Porton Down, Wiltshire, UK). TMD7 (acute myeloblastic leukemia with trilineage myelodysplasia) and TMD8 (diffuse large B-cell lymphoma) cell lines were established in our laboratory. Recombinant human EPHB4 and ephrin B2–Fc chimera were purchased from R&D Systems (Minneapolis, MN, USA), and a specific EPHB4 inhibitor NVP-BHG712 (14) was purchased from Selleckchem (Houston, TX, USA). The recombinant human NOTCH ligand protein delta-like 1 (DLL1) was provided by Dr. S. Sakano (Asahi Kasei Corporation, Fuji, Japan) (8).
Primer sequences (5’-3’) used for reverse transcription-polymerase chain reaction.
Short-term growth assay. The effects of ephrin B2, EPHB4, and NVP-BHG712 on short-term growth were examined using a colorimetric WST-1 proliferation assay. Cells (2-4×104 /well) were seeded in 96-well plates in 0.1 ml of RPMI-1640 medium (GIBCO, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum with increasing concentrations of ephrin B2 and NVP-BHG712. Cells were also seeded in 96-well plates pre-coated with EPHB4 (15) under the same conditions. After three to five days of culture, a solution of WST-1 and 1-methoxy-5-methylphenazium methylsulfate (Dojindo Laboratories, Kumamoto, Japan) was added to the wells. Cell proliferation was assessed by measuring optical density using an enzyme-linked immunosorbent assay (ELISA) reader. The Student's t-test was used to determine the statistical significance of the differences between the control and treated cells.
To examine cell differentiation, preparations of the cultured cells were made by Cytospin 3 (Shandon, Cheshire, UK), stained with Wright, and observed under a microscope.
Colony assay. Cells were plated at 2-5×102 cells/well in 96-well plates in 0.1 ml of MEM Alpha (GIBCO) containing 0.8% methylcellulose with 10% fetal bovine serum with or without ephrin B2. After seven days, colonies containing more than 20 cells were counted under an inverted microscope.
Reverse transcription-polymerase chain reaction (RT-PCR). The expression of the EPH and EFN genes was examined by RT-PCR. Total RNA was extracted, and first-strand cDNA was synthesized. The sequences were amplified by PCR using specific primers (Table I), separated by agarose gel electrophoresis, and stained with ethidium bromide. The regulatory effects of ephrin B2 and DLL1 on gene expression were examined by quantitative RT-PCR using a FastStart DNA Master SYBR Green I kit, LightCycler primer sets (Roche Diagnostics, Mannheim, Germany), and QuantiTect primers (QIAGEN, Hilden, Germany). The expression levels of each mRNA was normalized to that of β-actin (ACTB) mRNA.
Expression of EPH and EFN genes analyzed by qualitative reverse transcription-polymerase chain reaction.
Immunoblot analysis. The effects of ephrin B2 and EPHB4 on protein phosphorylation were examined by immunoblotting. Cells were treated with ephrin B2 or EPHB4 for 24 h, harvested, and lysed. The lysates were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with antibodies against phospho-tyrosine (4G10; Upstate Biotechnology, Lake Placid, NY, USA) and α-tubulin (Abcam, Cambridge, MA, USA) used as loading control.
Microarray analysis. The effect of ephrin B2 on gene expression was examined by two-color microarray analysis. Total RNA was extracted from cells cultured with or without 1 μg/ml of ephrin B2 for 24 h. Cy5-labeled and Cy3-labeled cDNA from each RNA sample was hybridized to an SurePrint G3 Human GE microarray 8×60K ver.2.0 (Agilent Technologies, Santa Clara, CA, USA) and analyzed using the Agilent Feature Extraction 10.7.3.1 software.
Results
Expression of the EPH and EFN genes. All analyzed cell lines expressed various combinations of the EPH and EFN genes (Table II). EPHA2, EPHA6, EPHA8, EPHB4, and EFNA4 were expressed in all cell lines; EPHB3 and EFNB2 were not detected in Jurkat and TMD8 cells, respectively, but expressed in all other cell lines.
Differential expression of representative genes in ephrin B2-stimulated AA cells analyzed by microarray.
Effects of ephrin B2 and EPHB4 on cell growth. The effects of ephrin B2 and EPHB4 on short-term growth and colony formation are presented in Figures 1 and 2. Ephrin B2 stimulation promoted the growth (Figure 1) and colony formation (Figure 2) of erythroid leukemia-derived AA cells, but had no significant effect on these parameters in the other cell lines (Figure 1 and data not shown). None of the cell lines responded to treatment with EPHB4. The analysis of cell morphology in the cytospin preparations did not detect changes in cell differentiation after the exposure to either ephrin B2 or EPHB4.
Effects of ephrin B2 and EPHB4 on protein phosphorylation. The immunoblot analysis of protein tyrosine phosphorylation in leukemia cells is shown in Figure 3. In AA cells, ephrin B2 stimulation promoted the phosphorylation of several proteins with approximate molecular weights of 60, 70, 90, 120 and 200 kDa, but did not affect the phosphorylation in other cell lines. The stimulatory effects of EPHB4 were not detected in any of the tested cells (data not shown).
Gene expression profile in ephrin B2-stimulated cells. To address the mechanism of ephrin B2 growth-promoting effects in AA cells, we examined gene expression profiles in these cells subjected to ephrin B2 treatment by microarray hybridization. The results revealed that ephrin B2 stimulation increased mRNA levels of cell growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B (FOSB) and v-src avian sarcoma viral oncogene homolog (SRC) while down-regulating the expression of the genes involved in cell fate determination such as SRY-box 18 (SOX18) and GATA binding protein 4 (GATA4) (Table III).
Relationship between EPH–ephrin and NOTCH signaling. To examine the crosstalk between EPH–ephrin and NOTCH signaling, we assessed the expression of genes affiliated with these pathways in cells stimulated with ephrin B2 or DLL1. In THP-1 cells, ephrin B2 up-regulated the expression of the DLL1 and HES1 genes by approximately 220 and 880%, respectively, while DLL1 caused a 260% increase but a 52% decrease in the levels of EPHB3 and EPHB4 genes, respectively. No significant changes were observed for the other cell lines.
Effects of EPHB4 inhibitor on cell growth. To further investigate the molecular mechanisms involved in EPH–ephrin signaling in leukemia cells, we examined the effects of an EPHB4 inhibitor, NVP-BHG712, on cell growth. Treatment with 5 to 25 nM of NVP-BHG712 did not significantly affect the proliferation of the tested leukemia cell lines (data not shown).
Discussion
In this study, we showed that all seven tested leukemia cell lines expressed mRNA for various EPH and EFN genes, suggesting the possible involvement of EPH–ephrin signaling in leukemia-related functional mechanisms. On the other hand, exogenous stimulation with the recombinant ephrin B2 increased proliferation in only one cell line, whereas EPHB4 did not have any effect. The limited effects of exogenous ephrin B2 and EPHB4 on leukemia cell growth in our experiments may be explained by the constitutive activation of multiple endogenous EPH receptors and ephrin ligands expressed by leukemia cell lines (Table II); however, this hypothesis requires further experimental support.
Effects of ephrin B2 and erythropoietin-producing hepatoma-amplified sequence receptors B4 (EPHB4) on the short-term growth of leukemia cell lines examined by WST-1 assay. The cells were cultured with the indicated concentrations of ephrin B2 or EPHB4 for three to five days. Cell growth was assessed by the optical density (OD) and is presented as the percentage of the mean OD value for control unstimulated cells. *p<0.05, significantly different from the control.
The only cell line responsive to ephrin B2 was AA, derived from pure erythroid leukemia cells. Given that ephrin B2 stimulation promotes erythropoiesis of normal hematopoietic cells (2), the effects of ephrin B2 might be specific to erythroid cells. Along with growth promotion, ephrin B2 also induced tyrosine phosphorylation of several protein species. Since EPH receptors are tyrosine kinases (1), this result suggests the possibility that exogenous ephrin B2 caused the activation of some EPH receptors. We also showed that ephrin B2 stimulation up-regulated the expression of growth-related genes FOSB and SRC. It is known that ephrins and EPHs activate bi-directional signaling (1); however, we were unable to show the existence of the reverse signal from EPH to ephrin in our experiments.
Effects of ephrin B2 on the colony formation by AA cells. The cells were cultured in MEM Alpha supplemented with methylcellulose and ephrin B2 for seven days, and the colonies containing more than 20 cells were counted. The data are shown as the mean colony number/well±standard deviation.
We also found that stimulation with exogenous ephrin B2 increased the levels of NOTCH-related genes; conversely, NOTCH ligand DLL1 affected the expression of the EPH genes in THP-1 cells. These findings suggest that in leukemia cells, the EPH–ephrin and NOTCH signaling pathways are interrelated; however, the biological significance of this relationship needs to be elucidated.
Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH–ephrin signaling may be involved in the pathology of leukemia. To obtain a clue to novel therapies targeting EPH–ephrin pathways against leukemia cells, we examined the effects of NVP-BHG712, a commercially available EPHB4 inhibitor, on the growth of leukemia cell lines. However, we did not observe any effects on the growth of leukemia cells, including ephrin B2-responsive AA cells. This might be because the constitutive activation of other EPH receptors compensates for the inhibition of EPHB4. Further investigation of the molecular mechanisms underlying EPH–ephrin signaling in leukemia cells should be performed in order to develop effective therapies targeting EPH–ephrin pathways against leukemia.
Effects of ephrin B2 on protein tyrosine phosphorylation. The cells were cultured with or without 1 μg/ml of ephrin B2 for 24 h. The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibody to phospho-tyrosine (antibody to α-tubulin was used to ensure equal loading).
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (no. 18690522).
- Received February 20, 2014.
- Revision received April 18, 2014.
- Accepted April 22, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
