Abstract
已知游離輻射主要造成細胞週期停滯於 G1與 G2 兩期. 目前的研究指出腫瘤抑制基因 p53 參與 G1 期停滯的調控; 現今的說法是細胞在遭受游離輻射逆境後引發p53蛋白質的累積與轉錄活性的上升, 進而活化 p21/CIP1/WAF1一G1 週期素依存性蛋白激脢活性的抑制蛋白-的表現, 因而造成G1 期的停滯. 另外, 已知 G2期週期素依存性蛋白激脢活性的缺失是導致G2 期停滯的主因. 由於游離輻射造成細胞週期停滯的主要下游訊息傳遞路徑所知較為清楚, 同時細胞週期又可運用流動細胞分析術予以簡便快速分析, 所以, 本實驗以游離輻射誘致細胞週期停滯的效應為模式,探討其中可能涉及的上游調控分子. DNA-PK 係由 Ku 和 DNA-PKcs 所組成,DNA-PK是一絲氨酸蘇氨酸蛋白激脢可 DNA 游離端點活化. Ku 是由 Ku70和 Ku80 所構成的異質二聚體. 具有 DNA 端點結合能力, 係DNA-PK 的調控單元. 已知 DNA雙股斷裂是游離輻射造成細胞死亡最主要的傷害,DNA-PK可以受游離端點活化的特性顯示其可能涉及細胞輻射逆境效應的調控.此外在試管實驗中證實 DNA-PK 可以磷酸化許多蛋白質, 包括: p53, c-Jun, c-Fos, SRF, Sp1 ...等. 目前的幾項發現顯示DNA-PK 可能涉及由游離輻射誘致p53相關效應: 首先, 游離輻射誘致p53蛋白的累積並不需要轉錄作用; 有些實驗結果也指出磷酸化作用可能涉及該現象的調控. 另外, 已知 DNA雙股斷裂是引發 p53相關效應的主要訊號, 同時又可以活化DNA-PK 的活性. 最後,人類 p53蛋白的 DNA-PK 磷酸化部位 (Ser15)之突變會導致p53抑制細胞生長的能力下降, 顯示 DNA-PK 可能涉及p53抑制細胞生長能力的調控.我們運用Ku80 反意訊息核糖核酸的表現以阻斷 Ku80蛋白質的合成, 進而造成 Ku或DNA-PK 整體缺失;藉此以研究在游離輻射誘致p53相關效應上所扮演的角色. 研究結果顯示Ku80反意訊息核糖核酸確實部份地減低特有的 DNA 端點結合能力,同時也增加細胞對游離輻射的敏感性; 但是, Ku80 反意訊息核糖核酸的表現對游離輻射誘致G1 與 G2期停滯, 並沒有顯著影響. 此結果顯示 Ku 或 DNA-PK 可能並不參與游離輻射誘致細胞週期停滯的調控.此外,我們也探討其它蛋白質磷酸化與去磷酸化作用涉及游離輻射誘致細胞週期停滯效應的調控. 我們運用已知的蛋白激脢與磷酸酯脢的抑制劑以研究他們對游離輻射誘致細胞週期停滯效應的影響. 我們初步的研究顯示蛋白質絲氨酸/ 酪氨酸與酪氨酸磷酸化作用在X-光誘致 G1 期停滯的調控上可能扮演重要角色.Mammalian cells response to DNA damage by blocking the cellcycle in G1 and G2 phases. Here we studyed the upstream signaltransduction pathways by which the cell recognizes andprocesses DNA damage to cell cycle regulatory mechinery. DNA-PKconsists of Ku and DNA-PKcs, and is a novel protein Ser/Thrkinase activated by DNA double-stranded breaks (DSBs) which arethe most detrimental DNA lesion caused by IR. Ku is aheterodimer (Ku70 and Ku80) and has the DNA end-binding (DEB)activity. DNA-PK can phosphorylate p53 in vitro. The mutationof phosphorylation site (Ser15) putatively by DNA-PK in humanp53 protein reduces its ability to inhibit cell growth,suggesting the involvement of DNA-PK in p53-dependent G1arrest. Here, by expression of anti-Ku80 antisense mRNA, westudied whether Ku or DNA-PK is the upstream signallingmolecule that leads to cell cycle G1 arrest by IR. As expected,expression of anti-Ku80 antisense mRNA decreased DEB acivity,and resulted in a radio- sensitive phenotype. However, thedeficiency of Ku80 expression did not decrease the extent of X-ray-mediated G1 and G2 arrest. Our results suggested that Ku orDNA-PK contributes to cellular radiosensitivity but notregulation of IR-mediated cell cycle alterations. We also askedwhether other protein phosphorylation and and dephosphorylationcontribute to regulation of IR-mediated cell cycle arrest.Inhibitors of protein kinases and phosphatase were employed tostudy their effects on cell cycle progression following X-irradiation. Both 6-DMAP and genistein effeciently abolished X-ray-meidated G1 arrest while did not disrupt normal cell cycleprogression through G1 phase. Both 6-DMAP and genistein did notinterfere with the p21/CIP1/WAF1 mRNA induction by X-ray. Ourpreliminary results suggested the important role for proteinSer/Thr phosphorylation in the modulation of X-ray- mediated G1arrest.