Abstract

Several studies including ours have suggested that lack of CD56 expression in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56 MM has not been well elucidated. In this study, we sorted out both CD56 and CD56+ fractions from MM cell lines or patients with MM, and investigated their different responsiveness to interleukin-6 (IL-6) or insulin like growth factor-I (IGF-I), and tried to clarify the course of action in cell cycle distribution. After stained with PE-CD56, CD56 and CD56+ fractions in KMS-21-BM and U-266 cell lines were isolated by the cell sorter, and cultured separately either in the presence or absence of IL-6 (2 ng/ml and 10 ng/ml, respectively). Although CD56 cells in both KMS-21-BM and U-266 cell lines responded significantly to IL-6 (P=0.001 and 0.009, respectively), CD56+ cells did not. Ki-67+ cells in CD56 KMS-21-BM cells, that were significantly fewer than that in CD56+ ones (P=0.0003), increased significantly upon 24-hour incubation with IL-6 (P<0.0001). Western blotting analysis showed that the level of cyclin D1 and p27 protein in CD56 KMS-21-BM cells were up- and down-regulated by IL-6 in a time dependent manner, respectively. IL-6 also brought phosphorylation of Akt (ser473) in the CD56 cells. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67+ cells in the CD56+ cells did not respond to IL-6. Although IGF-I did not increase Ki-67+ cells either in the CD56 and CD56+ cells from KMS-21-BM, anti-IGF-I mAb significantly reduced Ki-67+ cells only in the CD56+ cells (P=0.006). IGF-I up-regulated the level of cyclin D1 and phosphorylated Akt in CD56+ KMS-21-BM cells. LY294002 completely blocked these effects of IGF-I. Same results were obtained in the analysis of U-266 cell lines. The MM cells sorted from 17 patients with MM were also examined for CD56 and Ki-67 expression. Four and 13 patients were distributed to the CD56 and CD56+ group, respectively. These MM cells from the patients were cultured with or without IL-6 (10 ng/ml) or IGF-I (500ng/ml) for 24 hours. IL-6 increased the percentage of Ki-67+ cells in the CD56 group more than those in the CD56+ group (P=0.007). Although MM cells did not respond to IL-6, IGF-I significantly increased Ki-67+ cells in the CD56+ group (P=0.005). These results suggest that CD56 and CD56+ MM cells could be stimulated by different cytokines. We here found that CD56 MM cells were proliferated more than CD56+ MM cells in the presence of IL-6, and that this effect of IL-6 was mainly mediated by the activation of PI3-K/Akt pathway. In addition, our results suggest that IGF-I play an important role in the proliferation of CD56+ MM cells via PI3-K/Akt pathway.

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