Abstract
Telomerase, a specialized ribonucleoprotein complex that extends telomeres of eukaryotic chromosomes, is repressed in normal human somatic cells but is activated in most cancers and immortal cells, suggesting that telomerase activity plays an important role in carcinogenesis. Exactly how telomerase activity is regulated in human cells is not known and is a topic which is presently under intensive investigation. During the T lymphocyte development, the expression of telomerase activity is highly regulated. Mature T lymphocytes express low or undetectable telomerase activity. Activation of T lymphocytes stimulates cell proliferation and is accompanied by transient activation of telomerase activity. In this study, we employed T lymphocyte activation as a model system to investigate how telomerase activity is regulated in human cells.Peripheral blood mononuclear cells（PBMC）were used as the source of lymphocytes and phytohemagglutinin（PHA）was used as a mitogen to activate T lymphocytes. Following T cell activation, rapid expression of hTERT, the catalytic subunit of telomerase, is followed by the activation of telomerase activity. The peak expression occurred at 1~2 days for the hTERT and 2~3 days for telomerase activity. In the presence of PKC inhibitor（Go6976）, both the induced expression of hTERT and telomerase activity was inhibited completely, indicating that the transient activation of telomerase activity during T cell activation is regulated by PKC-dependent signal transduction to induce the expression of hTERT. To address which PKC isoform may participate in the regulation of telomerase during T cell activation, we investigated：（i）the levels of individual PKC isoforms during T cell activation, and（ii）the effect of ectopic expression of individual PKC isoform on the promoter activity of hTERT using a luciferase reporter plasmid（pBTdel-408）that contains the proximal region（-408 bp）of hTERT promoter. During T cell activation, the level of PKC bI decreased initially, but restored after 1~2 days. In contrast, the level of PKC z increased gradually and reached a plateau 2 days after PHA stimulation. The levels of PKC a, - d and - e was little changed during the activation process. Attempts to examine the effect of individual PKC isoform on the promoter activity of hTERT in primary T lymphocytes were unsuccessful because these primary cells were resistant to transfection. Therefore, this study was conducted with a T cell line（Jurkat）derived from an acute T cell leukemia patient. Cells were cotransfected with pBTdel-408 and plasmids that express wild type or constitutively activated mutant form of PKC isoform, and assayed for luciferase activity. Ectopic expression of wild type PKC a, - bI, - d, - e, - z or constitutively activated type PKC a or - e had no stimulatory effect on the promoter activity of hTERT. However, expression of constitutively active PKC bI, - d, or - z appeared to produce a slight stimulatory effect on the hTERT promoter. Taken together, these results suggest that the transient activation of telomerase activity during T lymphocyte activation is largely mediated by transcriptional activation of hTERT expression through PKC-dependent signal pathway. Our preliminary data implicate that the PKC bI, - d, and/or - z may be the PKC isoform(s) that functions in vivo for the activation of hTERT expression.考試委員審定書中文摘要英文摘要誌謝序論………………………………………………………………………1 端粒……………………………………………………………………1 端粒與老化……………………………………………………………2 端粒□…………………………………………………………………3 端粒□與癌化的關係…………………………………………………4 端粒□的調控…………………………………………………………5 蛋白激□C…………………………………………………………….7 PKC於淋巴球活化過程中對端粒□活性的調控……………………9材料與方法……………………………………………………………..11質體和細胞的培養……………………………………………….….11人類週邊血液單核球細胞的製備與培養……………….………….11Jurkat細胞的培養……………………………………………………11蛋白激□C質體……………………………………………………..12Luciferase reporter for hTERT promoter………….………………….12細胞蛋白質萃取………………………………………….………….13細胞萃取液中蛋白質濃度定量………………………….………….13端粒□活性的測定…………………...……………………………...13細胞RNA萃取……………….……………………………………..14細胞RNA濃度定量………….……………………………………..15反轉錄聚合□連鎖反應………………………………….………….15西方墨點法……………………...…………………………………...16DNA的轉殖…………………………………………………………17冷光酵素活性的測定…………………………………….………….17結果………………………………………………………….………….19Phytohemagglutinin（PHA）活化T淋巴球端粒□活性的可能機制…………………………………………………….………………19PKC在T淋巴球中活化端粒□活性的角色……………………….20不同PKC異構體於T淋巴球端粒□活化時的表現情形…………20不同PKC異構體於急性T淋巴球白血病細胞株中調控hTERT 啟動子的情形………………………………………………………….21討論………………………………………………………….………….24圖表………………………………………………………….………….29參考資料…………………………………………………….………….34附錄………………………………………………………….………….41