Abstract
我們用人類血癌細胞株來研究泰克索誘導細胞自發性死亡的機制。以20nM 的泰克索處理HL-60細胞1小時可以看見滯留在分裂狀態的細胞(M細胞)增 加，但之後並未造成死亡細胞增多；若用10nM的泰克索處理12小時，則數 小時後可見到死亡細胞逐漸增加，但M細胞增加並不明顯，由此可見泰克 索誘導之細胞自發性死亡不一定要在細胞分裂的狀態下才會產生。由同步 化細胞的實驗得知,在合成DNA過程中的細胞(S細胞)也對泰克索相當敏感 ，顯示細胞自發性死亡的機制可以由M和S兩種不同週期下啟動，這也代表 了兩種不同誘導細胞自發性死亡的機制。欲再深入探討這兩種機制的內含 ，我們改用U937細胞和各種蛋白質激脢的抑制劑(protein kinase inhibitor)，發現某些抑制劑(如PD98059)可以在處理下救回細胞，使其 免於死亡。而此種效果只適用於G1和S細胞，拯救不了已經進入M週期的細 胞。此外，我們也發現泰克索會加速細胞週期的進行，使之迅速進入M週 期而死亡。這種效果應該是來自p42/44 MAPK的短暫活化，因為抑制其上 游激脢MEK1/2可以阻止這個效果。另一方面，我們看到CaMKII脢的抑制劑 可以抑制M期細胞被泰克索誘導的自發性死亡，這個過程中我們發現泰克 索誘導之Bcl-2蛋白磷酸化與ICE的活化都會被抑制。此種抑制的效果，主 要在S細胞上看到，而M細胞上只看到ICE的活化被抑制，這些反應顯示在 不同週期下，細胞進行自發性死亡的路徑是不同的。有趣的是，我們也發 現在細胞進入M週期後，ICE會自動被活化，而在細胞脫離M週期後，這種 活化又自動終止。這個現象提供了泰克索誘導之M週期特異性細胞自發性 死亡另一個合理的解釋。 Induction of apoptosis by taxol (paclitaxel) was investigated on human leukemic HL-60 and U937 cells. In HL-60 cells, treatment with 20-nM taxol for 1 hr showed a mitotic block without a subsequent increase in apoptosis, and cells treated with 10 nM taxol for 12 hr showed an increase in apoptotic ratio within several hours without an increase in mitotic block. It shows that apoptosis does not necessarily result from mitotic block and these two phenomena can occur independently of each other. Studies from the synchronized cells indicated that the cells in S phase are also sensitive to the taxol, suggesting that the apoptotic programs can be initiated either in G2/M or S phases and which represent two different cytotoxic mechanisms of taxol. In U937 taxol-induced apoptosis initiated in G1 and S phases, but not for G2/M stage. Taxol accelerates the cell cycle progression of treated cells, which facilitate the onset of apoptosis in G2/M phase. This acceleration may result from transient activation of p42/44 MAPK, because inhibition of upstream MEK1/2 by PD98059 reversed this effect. In contrast, inhibitors of CaMKII are capable of protecting U937 cells from taxol-induced apoptosis, especial in G2/M stage. At the dosage that abolishes 80% of the apoptosis induced by taxol, KN-62 effectively inhibits both the Bcl-2 phosphorylation and ICE activation in cells at S phase; while only ICE activation can be suppressed for cells at G2/M stage. It is also found that ICE is spontaneously activated in G2/M stage but deactivated rapidly after cells exit the M phase. This phenomenon provides a rational explanation for the M phase-specific cytotoxicity of paclitaxel.