Proteomics Analytical Normal Liver Cells And Liver Cancer Cells, Dna Damage Repair Complex

Hepatoma is the most common malignancy in China and it is the second death cause induced by cancer. At present the pathogeny of hepatoma is not clear. It have been reported that defective DNA repair are one of the reasons that induce cancer. Double-strand breaks (DSBs) arise from exposure to IR and DNA damaging agents, but also from cell metabolism—in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D) J gene recombination. Defective DNA repair can predispose to cancer. Now the work on DSBs repair complex, especially its relations with cancer pathogenesy , has been the hot spot.With the development of new technology, such as Mass, two-dimensional electrophoresis, HPLC, we obtain a good opportunity to investigate the protein interaction from proteomic level. Meanwhile the amino acid-coded tagging (AACT) technique invented by our group is another good method to research the protein interaction. Here, we approached quantitative proteomics platform of mass spectrometry based amino acid-coded tagging (AACT) technique and an in vivo dual-tagging quantitative approach that integrated epitope-tagging which allows single affinity purification of the natural complexes formed at real-time to identify and compare the specific components of the DNA repair complex formed in normal liver cells and liver cancer cells from the same doner. Two critical DNA repair complex, H2AX complex and RAD52 complex, were chosen.The first part of the dissertation was a brief summary of recent research progresses on DNA repair signaling pathway, specially the DSBs pathways. We emphasized the components of the repair complex and the function of these components. Furtehermore, we discussed relations between DNA repair proteins and cancer. In the second part of this chapter, protein-protein interaction technologies were reviewed and summarized in the context of their respective strengths/weakness, especially emphasized the application high throughput approaches. In the end of this part, the purpose and significance of our study were introduced. The research work of this dissertation was composed of three parts. H2AX complex and RAD52 complex were focused respectively.(1) Proteomic dissection of H2AX complexH2AX was the one of the variants of Histone H2A. First identified in 1980 in human cells as an electrophoretic isoform of the core histone H2A , H2AX was subsequently sequenced in the late 80' s. It had been reported that the modification of core histones could promote the DNA repair. There are three H2A subfamilies: H2A1, H2A2, H2AZ, and H2AX. H2AX constitutes a major H2A species, and its levels vary from 2 - 25%. Accumulating evidence has shown that H2AX is a key factor in the repair of DNA double strand breaks (DSBs). H2AX is rapidly phosphorylated (γ-H2AX) following exposure of cells to IR or DNA damaging agents , which induces DSBs, and forms foci at the damage sites. It was proposed thatγ-H2AX may serve as a docking site for other DNA damage repair/signaling proteins to bind in the vicinity of DNA lesions. H2AX complex may be a major source of genome instability, and defend the cancer. To understand the regulatory mechanisms of this complex, we approached integrated AACT , epitope tagging , co-immunoprecipitation (Co-IP) and HPLC to globally investigate the specific components of the H2AX complex formed in normal liver cells (7701 cells)and liver cancer cells (7703 cells) from the same donor. Stably expressed flag-H2AX 7701 and 7703 cells were constructed as our research objects. The epitope tagging could increase the efficiency of IP and therefore enhance the identification possibilities of protein complex; at the same time, the quantitative method of MS-based AACT could effectively differentiate the specific protein partners from the non-specific ones. Two groups were set to learn the proteins in 7701 cells and 7703 cells respectively, and the third group to compare the difference of H2AX complex in 7701 cells and 7703 cells. As a result, 85 specific H2AX interaction proteins were identified in 7701 cells, and 145 specific H2AX interaction proteins were identified in 7703 cells. The comparison group got more proteins, including 93 upregulated proteins in 7701 cells and 69 upregulated proteins in 7703 cells. The identified proteins included known protein partners such as PRAP-1,KU70,histone H4, and many unknown interaction proteins, such as CFL, PPM1G and some ribosomal proteins. Parts of proteins were chosen to further confirm by Westhern blot and Confocal laser scanning. The results showed the consistency with the MS results. On the other hand, the composition and function of H2AX protein complex were compared between 7701 cells and 7703 cells. As a result, the identified interaction proteins involved in more physiological functions, including cell cycle, apoptosis, DNA metabolism, coenzyme metabolism , carbohydrate catabolism, etc. This result gave an evidence that cancer is complex progress, too. After that we detected the protein expression by stimulating the cells with bleomycin. The results showed many protein expression increasable, and confirm the proteins function. In our experiments we try to compare the amounts changing of same protein in different cell. Different mixing group and normalization control were used. The results showed many proteins have different binging in the amount. Because the 7701 cells and 7703 cells came from the same donor, it could reduce the false positive. At last we compare the protein expression in the hapatoma by immunohistochemical stain of some animal model sections. The results showed many protein' s expression increase with the cancer developing. We hope our results would give a potential biomarker for the digonosis and therapy of hepatoma.(2) Proteomic dissection of RAD52 complexRAD52 belongs to Rad52 epistasis group . Members of the Rad52 epistasis group, including Rad51, Rad52, DNA replication protein A (RPA), are important for homologous recombination. It was been reported that RAD52 had a role in many steps of DNA repair. The same strategy was approached in the experiment. we used integrated AACT , epitope tagging , co-immunoprecipitation (Co-IP) and HPLC to globally investigate the specific components of the RAD52 complex formed in normal liver cells (7701 cells)and liver cancer cells (7703 cells) from the same doner. Stably expressed c-Myc -RAD52 7701 and 7703 cells were constructed as our research objects. As a result, 89 specific RAD52 interaction proteins were identified in 7701 cells, and 129 specific RAD52 interaction proteins were identified in 7703 cells. Meanwhile we identified 60 proteins upregulated in 7701 cells, and 90 proteins upregulated in 7703 cells. With the function analysis, these protein involved in Carbohydrate catabolism, nucleotide metabolism, establishment of cellular localization, amino acid metabolism, protein metabolism, programmed cell death, Response to chemical stimulus, etc. comparing the results, we found some proteins working in anti-apoptosis in 7703 cells, not 7701 cells. At the same time some protein working in defending were found in 7701 cells, not 7703 cells. It suggested the different function on DNA repair between normal liver cells and liver cancer cells. DNA repair can predispose to maintain the genome stability and keep cell homeostasis in normal liver cells. On the other hand, the cancer cell can predispose to escape from the defect DNA repair.