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Abstract
Background:
The question as to whether stromal cell-derived factor-1 (SDF-1) stimulated myeloma cell growth has been controversial. We explored the possibility that SDF-1 may function as an autocrine growth factor of myeloma cells.
Methods:
CD138+ primary bone marrow myeloma cells and myeloma cell lines (RPMI8226, U266, and ARH77) were used. Chemotaxis in response to SDF-1 of the cells was analyzed using TranswellsTM. Cell proliferation was measured by a colorimetric assay. SDF-1 mRNA expression was analyzed by RT-PCR (reverse-transcription-polymerase chain reaction). SDF-1 and interleukin-6 (IL-6) receptor expression as well as signaling molecule phosphorylation levels, were examined using Western blot analysis. Concentrations of SDF-1 in the cell culture supernatants were measured by ELISA assay.
Results:
SDF-1 alone had no discernible effect on the proliferation of CD138+ primary myeloma cells or myeloma cell lines. In contrast, SDF-1 significantly enhanced IL-6-induced proliferation of these cells. SDF-1 up-regulated the expression of IL-6 receptor and enhanced phosphorylation of AKT in an additive manner with IL-6. Co-culture of the myeloma cells with umbilical vein endothelial cells over-expressing the SDF-1 gene revealed that SDF-1 played an important role in not only the migration of the cells underneath the stromal cells but also the proliferation of the cells in contact with stromal cells. All the myeloma cell lines expressed SDF-1 mRNA, and SDF-1 was detected in the culture supernatants of the cells. The G protein-coupled receptor inhibitor, pertussis toxin, inhibited the proliferation of these cells in suspension cultures.
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Fig. 1
Myeloma cells express functional CXCR4. (A) Flow cytometric analysis of the expression of CXCR4 on RPMI8226 cells, U266 cells, ARH77 cells, and CD138+ primary myeloma cells. ARH77 cells express CXCR4 minimally on the cell surface; however, analysis after permeabilization reveals CXCR4 in the cytoplasm of the majority of the cells. (B) Chemoattraction of myeloma cells induced by SDF-1 (100 ng/mL) over 4 hr measured using 24-well Transwells with 5-μm pores. Values represent the mean migration index (±SD) obtained from triplicate experiments for cell lines and those obtained from the experiments using five different specimens for CD138+ cells. ∗P＜0.05 compared with control.
Fig. 2
SDF-1 enhances the proliferation of myeloma cells in concert with IL-6. Cells were incubated in X-VIVO medium for 3 days in the absence or presence of SDF-1 (100ng/mL), IL-6 (20ng/mL), or both. (A) Proliferation of RPMI8226 cells. Data are the means and SD of the proliferation index of the cells from three independent experiments. (B) Proliferation of CD138+ primary myeloma cells. Data are the means and SD of the proliferation index of the cells from three independent experiments using three different specimens. ∗P＜0.05.
Fig. 3
Effects of IL-6 on the expression of CXCR4 and effect of SDF-1 on the expression of IL-6 receptor in myeloma cells. (A) IL-6 (20ng/mL) does not alter CXCR4 expression on the cell surface, as analyzed by flow cytometry. (B) In contrast, SDF-1 treatment (100ng/mL) for 24 hr up-regulates IL-6 receptor in RPMI8226 and U266 cells, as assessed by Western blot analysis. A representative result of two independent experiments is shown.
Fig. 4
Effects of SDF-1 and IL-6 on phosphorylation of AKT, ERK, and Stat3 in RPMI8226 cells. (A) Both SDF-1 (100ng/mL) and IL-6 (20ng/mL) increase the phsphorylation of AKT, and the combination enhances the phosphorylation of AKT compared to each cytokine. (B) SDF-1 modestly increases the phosphoryoation of ERK, and IL-6 strongly increases the phosphorylation of ERK. However, the combination does not enhance the phosphorylation. (C) SDF-1 does not cause the phosphorylation of Stat3 and does not enhance the phosphorylation induced by IL-6. A representative result of two independent experiments is shown.
Fig. 5
SDF-1 induces both the migration of myeloma cells beneath the stromal cells and the formation of cobblestone areas. (A) RPMI8226 cells were added onto MS-5 cell feeder layers in X-VIVO medium. The migration of the cells beneath the feeder layer was identified under an inverted microscope 24 hr later. (B) CD138+ primary myeloma cells were cultured in X-VIVO medium on established HUVEC layers, which had been transduced with the LacZ or SDF-1 gene using adenoviral vectors. A representative result of the 2-week culture with HUVECs transduced with the SDF-1 gene is shown. Many cobblestone areas are observed. (C) CD138+ primary myeloma cells (1×104 cells) were cultured on HUVEC layers transduced with the LacZ (AdeLacZ) or SDF-1 gene (AdeSDF-1). As indicated, the cells were pretreated with pertussis toxin (PTX; 200ng/mL) before co-culture. After 2 weeks, cobblestone areas were scored under a phase-contrast inverted microscope. Data are the means and SD of the numbers of cobblestone areas from three independent experiments using different specimens. ∗P＜0.05.
Fig. 6
Myeloma cells express SDF-1 mRNA and produce SDF-1. (A) The expression of the mRNA for SDF-1 and CXCR4 in myeloma cells, as analyzed by RT-PCR. (B) Western blot analysis for SDF-1 and CXCR4 in the cells. (C) SDF-1 concentrations in 3-day culture supernatants of the cells, as measured by ELISA.
Fig. 7
Pertussis toxin (PTX) inhibits the proliferation of myeloma cells. (A) RPMI8226 cells. (B) ARH77 cells. (C) U266 cells. The cells were cultured in X-VIVO medium for up to 4 days in the presence or absence of PTX (200ng/mL). Data are the means and SD of the numbers of cells from triplicate experiments. ∗P ＜0.05.
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