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Proteomic Analysis of p53-mediated Radiobiological Effects in Nasopharyngeal Carcinoma

Author: ShiHuiYing
Tutor: XiaoZhiQiang
School: Central South University
Course: Pathology and Pathophysiology
Keywords: Nasopharyngeal Radiobiology Proteomics P53 protein
CLC: R739.63
Type: Master's thesis
Year: 2008
Downloads: 79
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Abstract

Background and Purpose: NPC (Nasopharyngeal Carcinoma, NPC) is a common malignancy in southern China, and the major NPCs treatment for radiation treatment, but the overall effect is still not ideal, the 5-year survival rate is still hovering NPCs after radiotherapy at 40-50%. The p53 gene is a tumor suppressor gene involved in DNA damage repair, cell cycle regulation of DNA damage triggered run and apoptosis, tumor radiobiology effect mediated p53 tumor radiosensitivity closely related. Functional inactivation of the p53 gene is not have these features, resulting in lower tumor radiosensitivity, and even tumor radiation resistance. Studies have shown that more than 60% of the NPC organizations and almost 100% of the NPC cell lines p53 overexpression, and the NPC in the expression of p53 protein function may be abnormal or inactivation. NPC so far in the expression of p53 protein whether the impact of the radiation sensitivity of NPC is still unclear, p53 protein is mediated NPC radiation response mechanisms still need to be further clarified. Therefore, the observed expression of p53 protein radiosensitivity of NPC, looking for the p53-mediated radiotherapy sensitivity related protein, p53 involved in the NPC radiation response mechanism will help to clarify. Methods: (1) of p53 silencing human nasopharyngeal carcinoma cell line CNE2 radiobiology characteristics: stable silencing of p53 gene expression of NPC cells the strains CNE2sip53 and control cell lines CNE2/pSUPER object using colony survival analysis to detect in different radiation dose irradiation cell survival fraction (SF) and calculate the the radiobiology parameter D ₀, α, β, and radiation sensitive than (SER); MTT assay was used to detect the impact of radiation on cell growth; The staining detected radiation flow cytometry and Hochest33342 cell cycle and apoptosis; (2) proteomics technology to find and p53-mediated the CNE2 cell radiation sensitivity associated protein: two-dimensional gel electrophoresis (2-DE) the protein before and after the technical separation CNE2sip53, and CNE2/pSUPER cell radiation, PDQuest image analysis of differential protein spots around the the software identification CNE2sip53 CNE2/pSUPER cell radiation, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identification of differentially expressed proteins; Western blot validation of differences in protein of HSP70, the Tcp-1β, Annexin A2 and 14-3-3σ the CNE2sip53 well CNE2/pSUPER cell radiation around the expression level. Results: (1) The SF value higher than CNE2sip53 cells after irradiation cells SF CNE2/pSUPER; compared with CNE2/pSUPER cells, CNE2sip53 cells irradiated D ₀ values ??and SF2 values ??are increased, value of α, β value and SER were decreased; (2) the radiation induced CNE2sip53 apoptosis and cell proliferation inhibition was weaker than its role of CNE2/pSUPER cells; (3) the radiation induced CNE2/pSUPER cells were arrested in G1 phase arrest at the G2 phase induced CNE2sip53 cells; (4) to establish a two-dimensional gel electrophoresis the CNE2/pSUPER well CNE2sip53 cell radiation around the identification of 19 differentially expressed proteins around the CNE2/pSUPER cell radiation, mass spectrometry and CNE2sip53 cell radiation before and after the 15 differentially expressed proteins, Western blot validated 14-3-3σ, HSP70, Tcp-1β and Annexin A2 differential expression levels before and after the two cell radiation; (5) CNE2/pSUPER CNE2sip53 cells a comparative analysis of the similarities and differences of the differentially expressed proteins before and after radiation, identified 14 differentially expressed proteins, both Pyrophosphatase 1, Annexin A1 ,14-3-3 protein sigma, Proteasome (prosome macropain,) subunit, Prohibitin, Peroxiredoxin-2 Glutathionetransferase of GRP78 of precursor Cytokeratin 18, Ras-related protein, Glutathione transferase omega 1-1 NM23-H1 Calmodulin-relatedprotein and the Electron transfer flavoprotein subunit alpha; bioinformatics analysis showed that seven proteins that Annexin A1 ,14-3 -3 protein sigma Proteasomesubunit, Prohibitin, with Glutathione transferase, GRP78 of precursor and Cytokeratin 18 and p53 direct or indirect interaction between. Conclusions: (1) over-expression of p53 protein in NPC cells with biological functions involved in radiation-induced the NPC cell damage and apoptotic process; (2) using 2-DE technology to establish a cell radiation around the CNE2sip53 well CNE2/pSUPER. dimensional gel electrophoresis, mass spectrometry analysis of differentially expressed proteins were identified before and after the radiation before and after 19 CNE2/pSUPER cells differentially expressed protein and 15 CNE2sip53-cell radiation, provides valuable information for the study of NPC radiobiology; (3) found differences before and after the the 14 different CNE2/pSUPER and CNE2sip53 cell radiation protein, 14 protein may be associated with radiation and p53-mediated NPC NPC radiation response mechanism to provide an experimental basis to participate in order to clarify p53.

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