The effect of arsenic treatment on Aurora-A promoter methylation and screening for novel Ras-related proteins

• Main Authors: Yu-Te Chang, 張有德
• Other Authors: Hsiao-Sheng Liu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/95256085671458427401

碩士 === 國立成功大學 === 微生物及免疫學研究所 === 97 === Arsenic is an oncogenic metalloid. Long-term exposure to arsenic may lead to vascular diseases, cancers and death. Aurora-A is a mitotic kinase which is overexpressed in numerous cancer cell lines and clinical specimens. We previously unraveled that the incidence of Aurora-A protein overexpression in the bladder cancer patients of black-foot disease endemic areas is significantly higher than in other areas, suggesting that arsenic exposuremay induce Aurora-A overexpression. In the present study we elucidate whether the DNA methylation of CpG islands within Aurora-A promoter is involved in arsenic-induced Aurora-A overexpression. The cytosines of the 28 CpG dinucleotides on CpG islands within Aurora-A promoter are unmethylated in bladder and skin cancer cell lines as well as clinical skin cancer specimens determined by bisulfite sequencing PCR (BSP) and methylation-specific PCR (MSP). All together, long-term (4 weeks) exposure to arsenic (1 μM) of E7 cells doesn’t affect the methylation status of Aurora-A promoter. Arsenic-induced Aurora-A overexpression is not through demethylation of CpG islands within Aurora-A promoter. Ras is a well-known oncogene. Comparing the protein profile between cells with and without H-Ras12V overexpression total of 20 H-Ras-related proteins with approximate 2-fold expression difference were identified. By LC-MS/MS protein identification, one of them is phosphoglycerate mutase I (PGAM1), which may correlate with Ras activity. Furthermore, an E. coli strain that expresses inducible biotin-tagged H-Ras12V recombinant protein was established. This system will be used for the identification of novel Ras-interacted proteins, which will shed lights on the therapy against Ras-related cancers.