探討人類第二型拓樸酶在酸造成的DNA斷裂中所扮演之角色

Abstract

胞內酸化 (Intracellular acidification)被報導參與在器官局部缺血後再灌流 (ischemia/reperfusion) 所造成的器官傷害與細胞凋亡 (apoptosis)。之前研究也指出酸 (acidic pH) 可能造成DNA的損傷。然而，到目前為止，酸如何導致DNA的傷害以及酸化如何造成細胞死亡的分子機制還不清楚。在本篇論文中，我們利用細胞暴露在酸性的培養液中去模擬生理上當心肌梗塞或是中風時所造成的酸化現象。我們目前的研究清楚指出酸能直接造成DNA的斷裂而使DNA受到損害。由溫度及螯化物的實驗結果可推測出酸所造成的DNA 斷裂是需要酵素參與在其中的。有趣的是，這些由酸導致DNA片段的形成是可逆的；並且能被第二型DNA拓樸酶 (TOP2) 的抑制劑，ICRF-193，給中和抵消。由這些實驗結果顯示，TOP2可能參與在酸所引起的DNA傷害之中。與上述觀察到的結果一致，相對於TOP2表現正常的細胞 (HL-60)，在TOP2缺失的細胞 (HL-60/MX2) 中可看到由酸引起的DNA斷裂以及細胞死亡的現象明顯下降。另外，RNA干擾 (RNAi) 實驗結果顯示兩種hTOP2異構酶，hTOP2α和hTOP2β，在酸引起的DNA損傷及細胞死亡中分別扮演不同的角色。再者，不論是hTOP2α,hTOP2β RNAi-knockdown細胞株，或是TOP2β-/-剔除鼠胚胎纖維組織母細胞 (MEFs) 皆能抵抗由酸所造成的DNA傷害。總之，我們的研究結果推測出在ischemia發生時，hTOP2β可能在酸所引起的細胞凋亡及器官失能中扮演著重要的角色。我們也意外地觀察到較低的pH值會去抑制H9c2心肌細胞中由酸造成的DNA損害訊息的活化，因此對於在ischemia/reperfusion損害中所產生與pH相關的自相矛盾論點 (pH paradox) 提供了一個可能的解釋。

Intracellular acidification has been associated with apoptosis which has been attributed to mediate ischemia/reperfusion injuries and studies have also suggested that acidic pH might cause DNA damage. However, the molecular mechanisms underlying acidic pH induced-DNA damage and acidotic apoptosis remain unclear. In this thesis, cellular exposure to extracellular acidic pH (pHe) media was employed to simulate acidosis during heart failure and stroke. The present study indicates that acidic pHe can cause DNA damage by inducing DNA breakage directly. Temperature and chelator experiments suggested that acidic pHe-induced DNA breakage is enzymatic. Interestingly, the formation of these acidic pHe-induce DNA breakages is reversible and can be antagonized by a specific topoisomerase II (TOP2) catalytic inhibitor, ICRF-193. These results highly suggested the potential involvement of TOP2 in acidic pH induced-DNA damage. In agreement with above notion, hTOP2-deficient HL-60/MX2 cells, compared to parental HL-60 cells, exhibited a reduction in DNA breakage and apoptosis induced by acidic pHe. The differential role of hTOP2 isozymes, hTOP2a and hTOP2b, in acidic pH induced-DNA damage and apoptosis was revealed using RNA interference (RNAi)-specific knockdown cell lines. Either hTOP2a or 2b knockdown cell lines and TOP2b-/- knockout mouse embryo fibroblasts (MEFs) were resistant to acidic pH-induced DNA breakage. In sum, our study suggested that hTOP2b might play a more important role in acidotic apoptosis during ischemia. Unexpectedly, we have also observed that lower acidic pH inhibit DNA damage signaling and apoptotic pathways in H9c2 cardiomyocytes, thereby providing a potential explanation for the pH paradox in ischemia/reperfusion injuries.