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Viability Assays for Cells in Culture
Published on: January 20, 2014
[Proteomic analysis of G2/M arrest of HL-60 cells induced by MG132]
Jun-Jie Qian1, Wan-Tao Ying, Guo-Jing Sun
1Laboratory of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing, 100850, P. R. China.
Background & Objective:
Proteasome inhibitor, which can induce apoptosis in various tumor cells, is a kind of potential antitumor drug. This study was to identify the proteins involved in G(2)/M arrest of leukemia cell line HL-60 exposed to proteasome inhibitor MG132 by proteomic techniques.
Methods:
Flow cytometry was used to examine cell cycle of HL-60 cells exposed to 2.5 micromol/L MG132. Nuclear extracts of HL-60 cells were prepared, and the purity was detected by light microscopy and Western blot, and the differentially expressed protein spots were determined by two-dimensional gel electrophoresis and identified with MALDI-TOF-TOF/MS.
Results:
There was a distinct G(2)/M phase arrest before the apoptosis of HL-60 cells induced by 2.5 micromol/L MG132. Twenty-three differentially expressed protein spots were found between MG132-treated and control HL-60 cells; 8 nuclear proteins were identified by MALDI-TOF-TOF/MS analysis.
Conclusions:
The detected proteins, such as eIF5A and splicing factor, may be involved in regulation of G(2)/M arrest of HL-60 cells. These findings will be helpful for revealing molecular mechanisms of proteasome inhibitor-induced G(2)/M phase arrest and apoptosis of leukemia cell line.
Insights
Proteasome inhibitor MG132 causes G(2)/M arrest in leukemia cells before apoptosis. Proteomic analysis identified nuclear proteins like eIF5A and splicing factors involved in this cell cycle arrest mechanism.
Area of Science:
- Molecular Biology
- Cell Biology
- Proteomics
Background:
- Proteasome inhibitors are potential antitumor drugs that induce apoptosis.
- Leukemia cell line HL-60 is a model for studying drug-induced apoptosis.
Purpose of the Study:
- Identify proteins involved in G(2)/M arrest.
- Investigate the mechanism of proteasome inhibitor MG132 in leukemia cells.
Main Methods:
- Cell cycle analysis using flow cytometry.
- Proteomic analysis of nuclear extracts via 2D gel electrophoresis and MALDI-TOF-TOF/MS.
Main Results:
- MG132 induced distinct G(2)/M phase arrest in HL-60 cells.
- Identified 8 differentially expressed nuclear proteins, including eIF5A and splicing factors.
Conclusions:
- eIF5A and splicing factors may regulate MG132-induced G(2)/M arrest.
- Findings aid understanding of proteasome inhibitor mechanisms in leukemia.
