Mechanism of anti-sense APE/Ref-1 overexpression enhances cadmium-induced apoptosis

• Main Authors: Ai-Hsin Yen, 嚴愛鑫
• Other Authors: Jia-Ling Yang
• Format: Others
• Language: en_US
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/76883196184010380717

碩士 === 國立清華大學 === 生命科學系 === 88 === Abstract Apurinic/apyrimidinic endonuclease (APE), also termed as redox factor-1 (Ref-1), repairs the apurinic/apyrimidinic (AP) DNA damage and regulates the DNA binding activity of transcription factors such as AP-1, NF-kB, and p53 through the redox mechanism. Therefore, APE is critical for DNA repair and redox regulation upon oxidative or radiation damage to cells. In this study, we examined the effect of APE on the cytotoxicities induced by radiomimetic agents, including Cd, Cr(VI), As(III), MMS, and H2O2. Using retrovirus vector transfection, we established a stable cell line, APEas-CL3 that expression an antisense gene of APE/Ref-1 as well as the control counterpart Babe-CL3 cell line. Western blot analysis and APE repair activity assay showed that the APE protein levels decrease ~50% and the rate of repair AP site in APEas-CL3 cells was slower than that in Babe-CL3 cells. The APEas-CL3 cells were markedly more sensitive to the cytotoxicity induced by Cd than the Babe-CL3 cells, while these two cell lines have similar sensitivity to the cytotoxicity induced by Cr(VI), As(III), MMS, or H2O2. The Cd uptake ability and the rate of repair AP site in Cd-treated APEas-CL3 or Babe-CL3 cells were about the same, suggesting these events are not responsible for that APEas-CL3 cells are hypersensitive to Cd. Cd increased the hprt mutation frequency ~2-fold in APEas-CL3 cells but not in Babe-CL3 cells. Moreover, Cd markedly induced the formation of micronucleus in both cell lines at similar frequency. The apoptosis induced by Cd in APEas-CL3 cells was markedly higher than that induced in Babe-CL3 cells. Also, the pro-caspase 3 in Cd-treated APEas-CL3 cells cleavages more rapidly than Cd-treated Babe-CL3 cells, indicating Cd-induced apoptosis is mediated through the activation of caspase 3, which is enhanced by APE down regulation. Cd markedly activated the Akt survival signal pathway, however, the levels of its activation were similar in both APEas-CL3 and Babe-CL3 cells. Recent studies have shown that Cd-induced apoptosis is associated with mitogen-activated protein kinases, i.e., JNK and p38 activated by Cd transmit apoptotic signals, whereas ERK signal plays an opposing role (Chuang, Carcinogenesis 21(7), 2000). Analysis of activation of these protein kinases showed that the duration of p38 and JNK activated by Cd in APEas-CL3 cells is much longer than that in Babe-CL3 cells, while ERK re-activation after Cd withdrawal occurs in Babe-CL3 but not in APEas-CL3 cells. APE down regulation also results in a longer G2/M phase and faster destruction of cyclin B1 without affecting cellular growth rate. Together, APE may function in the G2/M checkpoint to ensure genome integrity and APE down regulation can enhance Cd-induced apoptosis through increased activation of caspase-3, enhanced persistency of JNK and p38 activities and reduced ERK re-activation.