Li Sun1, Hong-xiang Wang2, Xiao-jian Zhu1, Pin-hui Wu3, Wei-qun Chen4, Ping Zou1, Qiu-bai Li1, Zhi-chao Chen1. 1. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. 2. Department of Hematology, Wuhan Central Hospital, Wuhan 430022, China. 3. First Clinical College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. 4. Department of Central Laboratory, Wuhan Central Hospital, Wuhan 430022, China.
Abstract
AIM: To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from human myeloma cells and the implications for disease progression. METHODS: RPMI 8226, U266, and KM3 human myeloma cells were incubated in medium containing 10% (non-SD) or 1% fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF-MS, and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. RESULTS: Exposure of RPMI 8226 cells to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-κB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endothelial cells compared with non-SD MVs. CONCLUSION: SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.
AIM: To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from humanmyeloma cells and the implications for disease progression. METHODS:RPMI 8226, U266, and KM3 humanmyeloma cells were incubated in medium containing 10% (non-SD) or 1% fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF-MS, and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. RESULTS: Exposure of RPMI 8226 cells to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-κB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endothelial cells compared with non-SD MVs. CONCLUSION: SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in humanmyeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.
Authors: M Bernimoulin; E K Waters; M Foy; B M Steele; M Sullivan; H Falet; M T Walsh; N Barteneva; J-G Geng; J H Hartwig; P B Maguire; D D Wagner Journal: J Thromb Haemost Date: 2009-06 Impact factor: 5.824