Expression And Biological Behavior Of SDF-1/CXCR4 On Human Multiple Myeloma Cells

Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. MM cells appear to be derived from a pst germinal centre B cell as they express somatically mutated Ig heavy chain genes. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. It is possible that the bone marrow localization reflects the expression of chemotactic receptors on MM cells that directs their migration to the bone marrow.The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells.Part I. Plasma Levels of SDF-1 and Expression of SDF-1 Receptor onMultiply Myeloma Cells of Human Multiply Myeloma1. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1.XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that 瓼resh MM cells and MM cell lines expressed various levels of functional CXCR4ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[ (23.2?.08) %, PO.01]; DSDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons [(3489.23?51.63)pg/ml, (2818.57?97.79)pg/mL P<0,05 = ; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973 ?133)pg/ml, (2334.857 ?574.92), P=0.062]; ㏄lasma levels of PCL(4097.14 ?80.71) were significantly higher than those of healthy persons, P< 0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells.2. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules.3. We also found that SDF-1 could promote long-term proliferation of myeloma cells besides inducing migration of myeloma cells as IL-6 did. An up-regulation of autocrine IL-6(from 5.1pg/105 cells/24hours to 32.3 pg/105 cells/24hours) by XG1 cells was found in culture supernatant when SDF-1 was added in the culture system. The proliferation of myeloma cells induced by SDF-1 could be blocked by additional anti CXCR4 monoclonal antibody(12G5) or anti-IL-6 monoclonal antibody (B-E8); Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (y=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.4. Western-blot found that SDF-1 induced a transient up-regulation in the phophorylationof the p44/42 MAP kinase on XGs, but do not induced the phophorylation of the STATS on XGs. These study indicated that signal transduction of SDF-1/CXCR4 is through MAP kinase, but not STATS.5. We demonstrated that abnormal expression...