Original Article

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

Authors: Li Sun, Hong-xiang Wang, Xiao-jian Zhu, Pin-hui Wu, Wei-qun Chen, Ping Zou, Qiu-bai Li, Zhi-chao Chen
DOI: 10.1038/aps.2013.166

Abstract

Li SUN1, #, Hong-xiang WANG2, #, Xiao-jian ZHU1, Pin-hui WU3, Wei-qun CHEN4, Ping ZOU1, *, Qiu-bai LI1, *, Zhi-chao CHEN1
1Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of Hematology, Wuhan Central Hospital, Wuhan 430022, China; 3First Clinical College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 4Department of Central Laboratory, Wuhan Central Hospital, Wuhan 430022, China

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.


Keywords: multiple myeloma; microvesicles; nutrient deprivation; cell adaptation; NF-κB; ZNF224; SARM; COBL; two-dimensional gel electrophoresis

This research was supported by grants from the National Natural Science Foundation of China (81272624 and 81071943). We thank the Central Lab of Wuhan Central Hospital for the detection and analysis of MVs.
# These authors contributed equally to this study.
* To whom correspondence should be addressed.
E-mail qiubai.li@yahoo.com (Qiu-bai LI); zouping387@163.com (Ping ZOU)
Received 2013-08-10 Accepted 2013-10-08
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