| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === 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.
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