Proteomic Profiling of Arsenite-arrested Metaphase Cells

• Main Authors: Wan-Ching Won, 翁婉青
• Other Authors: Te-Chang Lee
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/97803115781641205991

碩士 === 國立中央大學 === 生命科學研究所 === 91 === Abstract Arsenic is a well-documented human carcinogen, primarily based on the evidence of clinical observation and epidemiological studies. Previous studies in our laboratory have shown that treatment of HeLa S3 cells with 5 μM arsenite leads to approximately 35% of the cells arrested in the mitotic metaphase. Treatment of arsenite-arrested mitotic metaphase cells with staurosporine (SSP, a protein kinase inhibitor) enforces them exiting from mitosis. In this thesis, experiments were conducted to ask how SSP enforces the mitotic exit. The microscopic structure of mitotic spindles and chromosomes were examined by immunofluorescent technique using anti-β-tubulin antibody and DAPI, and the content of Pds1 were analyzed by Western blot technique. The results show that treatment of arsenite-arrested mitotic cells with 50 nM SSP leads to degradation of Pds1 and increases in the frequency of cytokinesis. The proteins modified by SSP on arsenite arrested mitotic cells are analyzed by two-dimensional electrophoresis. These proteins are identified by MALDI-TOF technology. Several proteins are apparently modified by staurosporine treatment, for instance, carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein etc. Two-dimensional electrophoresis also detects differentially expressed proteins between the nocodazole and arsenite-arrested mitotic spindle fractions. These proteins are ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein. The involvement of these proteins in mitotic arrest warrants further investigation.