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研究生:楊偉磊
研究生(外文):Wei-Lei Yang
論文名稱:EffectofPKB/AktKinaseonCytotoxicityandMutagenicityInducedbyN-methyl-N'-nitro-N-nitrosoguanidine
論文名稱(外文):PKB/Akt激對MNNG誘發之細胞毒性與致突變性的影響
指導教授:楊嘉鈴
指導教授(外文):Jia-Ling Yang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:68
中文關鍵詞:PKB/Akt激N-甲基-N'-硝基-N-亞硝基亞氨甲二胺O6-甲基鳥糞嘌呤甲基轉移細胞毒性致突變性O6-甲基鳥糞嘌呤
外文關鍵詞:PKB/Akt KinaseN-methyl-N'-nitro-N-nitrosoguanidineMNNGO6-methylguanine-DNA-methyltransferaseMGMTCytotoxicityMutagenicityO6-methylguanineO6meG
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烷基化合物N-甲基-N'-硝基-N-亞硝基亞氨甲二胺(MNNG)會將核酸上鹼基烷基化造成多種核酸鍵結物,其中以O6-甲基鳥糞嘌呤(O6meG)會造成細胞毒性與致突變性。細胞中O6meG主要是由O6-烷基鳥糞嘌呤甲基轉移(MGMT)修補。先前的研究顯示烷基化合物亦會誘發訊號傳遞路徑,暗示著核酸損傷修補機制與訊號傳遞路徑可能具有關聯性。本論文利用轉殖人類MGMT基因(Mer+表現型)之中國倉鼠卵巢細胞株CHO-AT17-C3(AGT)與Mer-表現型之母細胞株CHO-neo9-C5(CHOM),研究烷基轉移酵素MGMT與MNNG誘發之訊號傳遞路徑的關係,以及探討被誘發之訊號路徑影響細胞及基因毒性的可能性。研究結果發現,CHOM 細胞相較於AGT細胞具有高量之內生性磷酸化蛋白激Akt表現,MNNG處理可誘發AGT細胞中Akt磷酸化,相對的CHOM細胞Akt磷酸化則被抑制。分析人類纖維母細胞HFW(Mer+)及GM0011(Mer-)中內生性Akt激活性及其受MNNG影響的情形也有與中國倉鼠卵巢細胞一致的結果。有絲抗原活化蛋白激(MAPKs)路徑中磷酸化ERK與p38含量與PTEN(PI3-激去磷酸)表達量於兩細胞株中皆不受MNNG影響。共同處理PI3-激抑制劑Wortmannin或LY294002加強MNNG誘發AGT細胞之細胞毒性,但減弱hprt基因突變率。使用去磷酸抑制劑Okadaic acid可明顯提高Akt活性,並降低CHOM之細胞毒性與基因突變率。由研究結果推測MGMT可能參與Mer+細胞內生性Akt活性之負調控,在MNNG的刺激下,MGMT須執行核酸修補,失去負調控Akt活性,因而促使Akt活性相對地上升。Mer-細胞中過低的MGMT使得內生性之Akt活性維持高量,在MNNG處理下,可能引發其他負調控機制如去磷酸,使Akt活性明顯降低。Akt訊號路徑參與保護MNNG對Mer+及Mer-細胞造成的細胞毒性。然而在Mer+細胞Akt活性可能誘發”錯誤傾向”修補或複製而增加基因突變率,在Mer-細胞補充Akt活性卻降低基因突變率,暗示Akt訊號路徑在調控MNNG誘發之核酸修補系統及突變生成過程扮演樞紐角色。

Alkylating agent N-methyl-N’-nitro-N-nitroso-guanidine (MNNG) forms a structurally diverse population of alkyl-DNA adducts. The O6-methylguanine induced by MNNG is the most significant adduct causing cytotoxicity and mutagenicity. This adduct is repaired by O6-methylguanine-DNA methyltransferase (MGMT) in cells. Previous studies have indicated that alkylating agents can also induce signal transduction pathways, suggesting that signal transduction pathways may be associated with DNA repair systems. We have adopted the MGMT highly expressed Chinese hamster ovary cells (phenotype Mer+, AGT) and the MGMT deficient parental cells (phenotype Mer-, CHOM) to investigate whether MGMT is involved in the regulation of signaling pathways induced by MNNG. We have also investigated the roles of MNNG-induced signals in cytotoxicity and genotoxicity. CHOM cells exhibit higher levels of endogenous phospho-Akt than AGT cells. MNNG significantly induced phospho-Akt in AGT cells, conversely it decreased the levels of phospho-Akt by in CHOM cells. These phenomena are also observed in Mer+ and Mer- human fibroblasts HFW and GM0011, respectively. MNNG did not elicit ERK and p38 MAPK signals nor did it alter the expression of PTEN, a PI3 kinase phosphatase, in both AGT and CHOM cells. Co- administrating Wortmannin or LY294002, PI3-kinase inhibitors, increased cytotoxicity and decreased mutagenicity induced by MNNG in AGT cells. Okadaic acid, a protein phosphatase inhibitor, markedly elevated the levels of phospho-Akt, and decreased cytotoxicity and mutagenicity induced by MNNG in CHOM cells. The results suggest that in Mer+ cells the activity of Akt may be negatively regulated by MGMT, which must conduct DNA repair upon MNNG exposure and thereby leave the Akt negative control complex to allow increase of Akt activity. In Mer- cells, low MGMT enables the endogenous Akt activity maintained at high levels, which may be decreased by other negative control mechanism such as phosphatase elicited upon MNNG exposure. Akt signal protects MNNG-induced cytotoxicity in both Mer+ and Mer- cells. However, in Mer+ cells Akt activity may elicit "error-prone" repair or replication pathways and subsequently increase mutation frequency. Conversely, supplement Akt activity can decrease mutagenesis in Mer- cells. The results suggest that Akt signal transduction pathway is a key regulator in controlling DNA repair, replication and mutagenesis in MNNG-treated cells.

中文摘要………………………………………………….. Ⅰ
英文摘要……………………………………….…………. Ⅲ
中英名詞對照………………………………………………. 1
壹、 文獻回顧……………………………………………... 3
一. 硝基胺化合物………………………………………3
二. 硝基胺化合物造成核酸傷害之機制……………………3
三. 硝基胺化合物之基因毒性與致突變性………………….4
四. 細胞中修補核酸鍵結物O6meG之機制……………….. 5
五. 錯誤配對修補機制與細胞程序凋亡之誘發…………….. 7
六. 烷基化合物誘發生物訊號傳遞路徑對細胞生理之影響…...8
七. P13-激/Akt訊號傳遞路徑………………………...10
八. 結語……………………………………………...17
貳、 前言………………………………………………….18
參、 材料與方法…………………………………………….20
一. 材料與化學藥品………………………………….. 20
二. 細胞來源與培養………………………………….. 20
三. 細胞聚落形成能力分析……………………………. 21
四. 基因致突變分析………………………………….. 22
五. 西方墨點法……………………………………….23
六. 數據分析………………………………………… 25
肆、 結果………………………………………………….26
伍、 討論………………………………………………….33
陸、 參考文獻………………………………………………37
柒、 圖表………………………………………………….55

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