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Author: 邱建智
chien-chih, chiu
Thesis Title: 抑制人類非小細胞肺癌細胞生長之模式
Establishment of tumor suppressing models of human non-small cell lung cancer cells
Advisor: 方剛
Fang, Kang
Degree: 博士
Doctor
Department: 生命科學系
Department of Life Science
Thesis Publication Year: 2004
Academic Year: 92
Language: 中文
Number of pages: 135
Keywords (in Chinese): GCV細胞凋亡人類非小細胞肺癌VP-16老化延滯性細胞死亡p53
Keywords (in English): GCV, apoptosis, human non-small-cell-lung-cancer, VP-16, senescence, delayed-apoptosis, p53
Thesis Type: Academic thesis/ dissertation
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  • 肺癌在台灣為一高度致死率的癌症,尤其以非小細胞肺癌(non-small cell lung cancer, NSCLC)患者佔所有肺癌多數,而非小細胞肺癌的傳統治療方式主要以手術及化療為主。由於此類癌細胞對許多藥物都具有抗藥性,因此對非小細胞肺癌的診斷及預後結果都不佳。有鑑於此,實驗主要目的即欲瞭解非小細胞肺癌細胞對化療藥物處理後的細胞內傳導機制,另外也試圖利用基因治療的方式,為非小細胞肺癌提供一個新的治療方向。因此論文的方向主要分為三部分:第一部分的實驗內容主要對帶有外源性(exogenous)第一型皰疹病毒胸腺嘧啶激素(HSV-tk)的非小細胞肺癌細胞株(CL-1)的細胞凋亡與週期蛋白(cyclin)的活化調控進行探討。實驗結果發現,經過guanosine 的類似物ganciclovir (GCV)誘導之後,帶有HSV-tk基因的非小細胞肺癌生長會受到顯著的抑制,但卻不會影響周圍的正常細胞。實驗也證明此生長抑制效果可藉由旁觀者效應(bystander effect)影響週邊的肺癌細胞,藉此可擴大HSV-tk/GCV治療的效果,這種抑制效應也可藉動物試驗(xenograft assay)上證實有顯著的效果。實驗結果發現GCV誘導的CL-1細胞週期停滯於S-G2/M時期,顯示細胞內DNA的合成受到阻礙,而無法通過細胞週期,致使細胞分裂停滯。進一步分析發現GCV能誘導CL-1細胞產生細胞凋亡(apoptosis)。西方轉漬試驗的結果則發現G2週期相關的週期素cyclin A、cyclin B1蛋白以GCV誘導後,有明顯的累積,推測是導致細胞週期停滯的原因。另外也發現細胞凋亡因子cycochrome c於細胞質累積,而且caspase-9、caspase-3都被活化,因此證實HSV-tk/GCV誘導CL-1細胞凋亡主要是經由粒線體釋放cytochrome c途徑所產生的。
    論文的第二部分則是以topoisomerase IIα抑制劑VP-16誘導p53基因缺失的非小細胞肺癌細胞株H1299,並探討H1299細胞對VP-16的敏感性及細胞反應。研究結果顯示低劑量VP-16可抑制p53缺失的非小細胞肺癌細胞株H1299細胞生長,受刺激的細胞週期停滯於G2時期,隨後引發細胞的凋亡。以西方轉漬試驗也發現VP-16所誘發的細胞凋亡過程中,cytochrome c會自粒線體釋出,並且也發現caspase酵素群中的pro-caspase-9被切割。值得注意的是VP-16誘導H1299細胞後並未引起caspase-3的活化,但是凋亡執行者的caspase-7卻證實被切割活化。綜合以上結果,推測VP-16所誘導的H1299細胞凋亡是經由cytochrome c的途徑引起的,並且細胞凋亡的產生與caspase-9和caspase-7的活化具有直接的關聯性。
    論文的第三部份則是探討p53突變的NSCLC細胞H1437經VP-16處理之後,其細胞內的反應機制。實驗顯示VP-16處理後的H1437細胞表現出非傳統(atypical)的細胞凋亡。在VP-16誘導6天之後才啟動細胞凋亡。此種延滯性細胞凋亡(delayed-apoptosis)啟動前,會先造成細胞分裂停滯、端粒酶活性消失及內小體的半乳糖酶(endosomal galactosidase, pH 6)被誘導大量表現等細胞老化特有的表型(phenotype)。同時,以VP-16處理的細胞,發現腫瘤抑制基因p16及p21基因被誘導表現,且隨著細胞週期調節因子cyclin A與cyclin B1的持續累積而導致細胞停滯在G2/M週期。此實驗結果推論VP-16導致H1437細胞先後啟動老化機制,再造成細胞凋亡。

    Lung cancer is the leading cause of death in Taiwan, and the non-small cell lung cancer (NSCLC) represents 80% of the total lung cancer cases. New therapeutic paradigms are constantly pursued and improved. Thus, in this thesis, gene therapy using suicide gene HSV-tk/GCV strategy was evaluated in NSCLC cells. On the other hand, the molecular pathway in NSCLC cells induced by chemotherapeutic agent, VP-16 was also discussed in-depth in this study.
    In the part I of thesis, NSCLC cells were transfected with a recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extension of the bystander effect were associated with the expression level of HSV-tk in stable transfectants. Western blot was used to analyze the release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose) polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B1 before committing to apoptosis cell death. These findings demonstrated that the HSV-tk/GCV system effectively inhibited the proliferation of NSCLC cells in vitro and in vivo through induction of apoptosis, therefore providing a rationale for further development.
    In the part II of thesis, we found that the dynamic changes occurring in NSCLC, H1299 (null-p53) cell populations during protracted VP-16 (DNA-topoisomerase II inhibitor) exposure. Flowcytometry was used to analyze changes in cell cycle progression in H1299 cells, and showed a significant decrease in G1 phase cells with a concurrent increase in G2/M phase cells following VP-16 treatment. Result of western blotting showed cytochrome c releasing from mitochrondria occurred in early stage of apoptosis. Activation of caspase-7 was detected, while the cleavage of caspase-3 was not seen. This work also showed that an immediate accumulation of cell cycle modulators, cyclin A and B1 was visualized during VP-16-sensitized cell arrest at G2/M-phase. Whereas upregulation of p21waf/cip prevented cells to overcome mitotic arrest before entering programmed cell death. In addition, the presence of caspase specific inhibitor abrogated caspase activation as well as VP-16-induced apoptotic phenotype. The finding of apoptosis mediated by VP-16 in subsets of human NSCLC cells provides an opportunity for targeted therapy for cancers caused by p53 deletion.
    In the part III of thesis, we have identified a pathway leading to VP-16-induced dose-dependent cell death in highly metastatic and tumorigenic NSCLC, H1437 cells with mutant p53. It was found that H1437 cells exposing to VP-16 was susceptible to cell proliferation and the effect proved irreversible both in vivo and in vitro. The inhibition of cell proliferation was triggered by senescence events exerted by low-dosage VP-16. In addition, H1437 cells underwent growth arrest at G2/M-phases prior to emergence of sub-G0/G1 cells. To further address the pathway in VP-16-induced H1437 cells, we have observed transient elevation of cyclin-dependent kinase inhibitor, p21CIPI/WAF1 and tumor suppressor, p16INK. At the meaning time, persistent escalation of endogenous cell cycle modulators, cyclin A and B1 signifantly induced DNA repair upon DNA damage before cell death. H1437 cells underwent apoptosis triggered by VP-16 at day 6 of exposure and were detected by annexin-V assay. Thereby, low-dosage VP-16 can be an effective agent in treating human NSCLC cells with mutated p53 genotype through a delayed-apoptotic pathway

    目錄 中文總摘要……………………………………………………3-5 英文總摘要……………………………………..…..…………6-7 第一部分: Transfer of herpes simplex virus-thymidine kinase cDNA in human non-small cell lung cancer cells conferred caspase-activated apoptosis upon ganciclovir sensitization ………………………………………………………………………8-51 前言………………………………………………………………….9-12 研究目的……………………………………………………………13 材料方法……………………………………………………………14-30 結果………………………………………………………………….31-35 討論………………………………………………………………….36-39 總結…………………………………………………………40 圖表………………………………………………………………….41-51 第二部分: Etoposide (VP-16)-induced apoptosis through caspase activation in human non-small cell lung cancer cells lacking endogenous p53 ………………………………………………………………………52-88 前言…………………………………………………………………53-55 研究目的……………………………………………………………56 材料方法……………………………………………………..…54-62 結果………………………………………………………………….63-69討論………………………………………………………………….70-75 總結….…………………………………………………………….76 圖表………………………………………………………………….77-88 第三部分: Etoposide (VP-16) triggers delayed-type apoptosis in human non-small-lung cancer cells with mutated p53 ……………………………………………………………………89-122 前言………………………………………………………90-93 材料方法………………………………………………………….94-102 結果…………………………………………………………….…103-107 討論……………………………………………………………….108-113 圖表………………………………………………………….….114-122 參考文獻………………………………………………………..123-134

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