亞砷酸鈉誘引有絲分裂期CGL-2細胞死亡機制之研究; The mechanism of arsenite-induced mitotic death in CGL-2 cells

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题名:	亞砷酸鈉誘引有絲分裂期CGL-2細胞死亡機制之研究;The mechanism of arsenite-induced mitotic death in CGL-2 cells
作者:	劉芳君;Fang-Chun Liu
贡献者:	生命科學研究所
关键词:	砷;有絲分裂;mitosis;arsenite
日期:	2006-07-03
上传时间:	2009-09-22 10:19:18 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	砷化物會造成許多腫瘤細胞進行不正常的有絲分裂，使細胞停滯在有絲分裂期，進而促使細胞走向細胞凋亡。本研究探討亞砷酸鈉造成CGL-2細胞停滯在有絲分裂期後進行細胞凋亡的機制。為了解亞砷酸鈉如何引起細胞停滯在有絲分裂期與啟動細胞凋亡，利用西方點墨法觀察有絲分裂管制點和細胞凋亡的蛋白質表現，結果顯示亞砷酸鈉在短時間內就會造成有絲分裂停滯，有絲分裂管制點活化，之後參與粒線體相關細胞凋亡的蛋白開始活化，並發生凋亡。將短時間與長時間停滯在分裂期的細胞做比較，發現透過粒線體產生的細胞凋亡在長時間的停滯後才明顯發生，並伴隨Bax構形改變、粒線體膜電位喪失與caspase活化；而自由基的產生在短時間停滯的細胞就被誘導，但卻沒有顯著的細胞凋亡的發生，且非分裂期的細胞並沒有明顯自由基的產生。這些結果說明，亞砷酸鈉造成細胞停滯在有絲分裂期後會誘導自由基產生，進而誘發粒線體相關的細胞凋亡。JNK和p38在亞砷酸鈉誘發的分裂期停滯及細胞凋亡的過程中被明顯磷酸化，顯示它們可能參與砷導致分裂期細胞凋亡的過程。另一方面，利用二維電泳發現STI1在亞砷酸鈉停滯在分裂期的細胞中有較高的表現量。除此之外，為了探討中心體倍增的機制，利用 g-tubulin做免疫沉澱，發現許多與 g-tubulin 結合的蛋白在亞砷酸鈉停滯在分裂期的細胞中表現較多，可能和砷引起的中心體倍增有關。本實驗證實亞砷酸鈉使細胞停滯在有絲分裂，造成有絲分裂管制點的活化，引發自由基的產生或累積，並可能透過JNK、p38、STI1和一些與中心體倍增相關的蛋白使長時間停滯在有絲分裂的細胞走向粒線體相關的細胞凋亡。 Arsenite can induce mitotic abnormalities, mitotic arrest, and subsequently lead to mitosis-mediated apoptosis in a variety of cancer cell lines. Knowledge of the mechanism of mitotic death can help realizing the toxicity of arsenite. In the present study, we confirmed the effects of arsenite on mitotic arrest and mitotic death in CGL-2 cells. To dissect how cell death was initiated in these mitotic arrested cells, the expression status of several mitotic checkpoint proteins and apoptotic proteins during arsenite-induced mitotic arrest were examined by Western blot analysis. The results showed that arsenite induced the activation of mitotic checkpoint. The apoptotic proteins were activated after protracted activation of the mitotic checkpoint. In addition, change of Bax conformation, loss of mitochondria membrane potential, and initiation of apoptosis was significantly induced in the long-term arsenite-arrested mitotic cells. Furthermore, ROS generation was noticed in short-term and long-term arsenite-arrested mitotic cells but not in interphase cells. These results indicated that ROS was generated after arsenite-induced mitotic arrest and mitochondria-mediated apoptosis was preceded with ROS generation. JNK and p38 were phosphorylated during arsenite-induced mitotic arrest and mitotic death indicating the activation of JNK and p38 might be involved in arsenite-induced mitotic arrest and mitotic death. On the other hand, 2D electrophoresis analysis revealed that the expression of stress-induced-phosphoprotein 1 (STI1) was higher in arsenite-arrested mitotic cells than in normal mitotic cells. Analysis of centrosome-associated proteins in normal or arsenite-arrested mitotic cells by immunoprecipitation with g-tubulin antibodies revealed that the expression of several proteins was higher in arsenite-arrested mitotic cells than in normal mitotic cells. These proteins might be involved in arsenite- induced centrosome amplification. Our results strongly suggested that inappropriate mitosis induced by arsenite could lead to the activation of mitotic checkpoint and the generation of ROS. The prolonged activation of mitotic checkpoint could lead to mitochondria-mediated apoptosis probably through JNK, p38, STI1, and some centrosome amplification-associated proteins.
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