The Signaling Pathways of DNA Damager MNNG- Induced Necrotic Cell Death
| Autor: | Ling-Ya Chiu, 邱鈴雅 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 Alkylating agent N-methyl-N’-nitro-N’-nitrosoguanidine (MNNG) can cause excess DNA strands break then leads to PARP-1 overactivation and necrotic cell death. However, the regulatory mechanism of PARP-1 activation in detail relating to identified necrotic features, such as reactive oxygen species (ROS) production, calcium elevation, ATP depletion and JNK activation, have not been fully understood. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to study the signaling pathways of PARP-1-mediated necrosis. We found that MNNG treatment can induce cell necrosis as assessed by the increased uptake of propidium iodide, and no protective ability of pan-caspase inhibitor ZVAD. MNNG induced cell death is featured by the rapid PARP-1 activation, JNK activation, biphasic ROS production, intracellular calcium increase and sustained ATP depletion. Results indicate that the early ROS production which occurred at 1 min and peaked at 5-15 min after MNNG treatment is resulting from NADPH oxidase, as DPI is able to inhibit such response. In contrast, the late phase of ROS production which occurred after 30 min and time-dependently increased up to 6 hr after MNNG treatment was generated by mitochondria. When treating cells with antioxidant NAC, all the phenomena caused by MNNG were abrogated. Studies with calcium chelator BAPTA/AM suggest that the rapid calcium rise, an event downstream of early ROS production, is involved in PARP-1 and JNK activation. Moreover, PARP-1 inhibitor (3AB) is able to reduce MNNG-induced PARP-1 activation, ATP depletion, late phase ROS production, calcium elevation, and cell death, but cannot affect the rapid JNK activation. On the other hands, studies with JNK inhibitor SP600125 indicate that JNK is involved in the late phase of ROS production. When comparing MNNG induced events in WT and RIP1-/- MEFs, our results indicate the necessity of RIP1 for mediating the sustained ROS production, calcium increase, as well as ATP depletion. Taken together, we have clarified the signaling crosstalk between PARP-1, ROS, calcium, JNK and RIP1 in MNNG elicited necrosis of MEFs. These results provide not only new insight into necrotic regulating mechanisms, but also into clinical benefits to patients in a variety of pathophysiological conditions associated with tissue necrosis. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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