Abstract
增生細胞核抗原(Proliferating Cell Nuclear Antigen)的功能為 DNA聚合酵素delta 和epsilon的輔助蛋白.當靜止細胞受生長因子刺激進 入細胞週期時,增生細胞核抗原mRNA的水平會上升,並於G1/S期間達到最高 點.本研究是利用穩定轉染作用(stable transfection)將大老鼠增生細胞 核抗原啟動子送入中國倉鼠卵巢(CHO.K1)細胞中選殖穩定轉染株,將之稱 為d693-pCAT.K1細胞,藉著其控制氯黴素乙醯基轉移酵素( Chloramphenicol acetyltransferase; CAT)的表現來研究大老鼠增生細 胞核抗原啟動子的特性.本研究印證以前瞬間(transient)轉染實驗的觀 察,即大老鼠增生細胞核抗原啟動子對血清刺激具有反應.以此為基礎探討 以紫外線照射穩定轉染株細胞,觀察大老鼠增生細胞核抗原啟動子活性之 表現,實驗結果顯示在指數生長期,G0期和G1晚期的細胞在受紫外線照射 後,大老鼠增生細胞核抗原啟動子會被活化.初步實驗結果顯示大老鼠增生 細胞核抗原啟動子被紫外線活化的可能位置在-70到+125的區域範圍內.另 外,研究p53和E2F對大老鼠增生細胞核抗原啟動子之影響,結果顯示p53和 E2F對大老鼠增生細胞核抗原都具有抑制效果,故紫外線對大老鼠增生細胞 核抗原啟動子活性活化的效應不可能是因為p53和E2F的緣故. Proliferating Cell Nuclear Antigen (PCNA) can function as an auxiliary factor of DNA polymerase delta and epsilon, is required for DNA synthesis andDNA repair. After serum stimulation of quiescent cells, the level of PCNA mRNAincrease and reaches its peak before onset of DNA synthesis. In the present study, a rat PCNA promoter with chloramphicol acetyltransferase (CAT) as reportwas stably transfected into CHO.K1 cells. The promoter named d693 containingsequences between -693 and +125 in reference to the transcription initiation aite has been previously shown to be serum responsive in transient expression study. The CAT activity in the stable transfectants named d693-pCAT.K1 cells also display the same character of serum-responsiveness. In addition, UV irradiation on d693-pCAT.K1 cells induced rat PCNA promoter activity in dose-dependence manner. Such UV inducibility was seen with quiescent cells and growing cells. Furthermore, the promoter region of rat PCNA promoter that isresponsible for UV induction are delimited within the region from -70 to +125,which contains an AP-1 and ATF/CRE site and has been shown to be serum responsive and E1A oncoprotein responsive in the previous studies. Furthermore,the UV inducibility of PCNA promoter dose not require the activities of p53 and E2F, as both p53 and E2F inhibited PCNA promoter activity when they were overexpressed in d693-pCAT.K1 cells. Thus, UV inducibility of the rat PCNA promoter dose not require p53 and E2F.