tailieunhanh - Prevention of hydrogen peroxide and cisplatin induced apoptosis by intracellular catalase overexpression
Apoptosis induced by hydrogen peroxide (H2O2) was investigated in Chinese hamsterovarian (CHO) cells, and compared to apoptosis induced by cisplatin (25 µM), a well-established apoptosis-inducing agent. | Turk J Biol 24 (2000) 685–696 © TÜBİTAK Prevention of Hydrogen Peroxide and Cisplatin Induced Apoptosis by Intracellular Catalase Overexpression H. Barbaros ORAL Department of Microbiology and Infectious Diseases, Immunology Unit, Uludağ University, School of Medicine, Bursa-TURKEY Adrew . GEORGE Department of Immunology, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London-ENGLAND Dorian O. HASKARD Department of Cardiovascular Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London-ENGLAND Received: Abstract: Apoptosis induced by hydrogen peroxide (H2O2) was investigated in Chinese hamster ovarian (CHO) cells, and compared to apoptosis induced by cisplatin (25 µM), a well-established apoptosis-inducing agent. In preliminary experiments, CHO cells were treated with low concentrations of H2O2 (250-500 µM) continuously or for 3 hours, followed by washing and further incubation in complete growth medium for 1-10 days. Fluorescence microscopy of acridine orange-stained cells indicated morphological changes of apoptosis. Similar changes were observed in cisplatin-treated cells. DNA-laddering also revealed that DNA fragmentation occurred in both adherent and non-adherent cells by day 2. To prevent H2O2-mediated apoptosis, the catalase gene was isolated, subcloned into a mammalian expression vector (pCDM8) and transfected into CHO cells. Transiently transfected cells contained approximately 8-fold increased catalase levels. Overexpression of intracellular catalase provided protection for the CHO cells from H2O2 as measured by a tetrazolium-formazan reduction assay. Furthermore, cisplatin-mediated apoptosis was prevented by intracellular catalase overexpression, indicating that this cytotoxic drug acts at least in part through an H2O2-dependent pathway. Key Words: Apoptosis, catalase, cisplatin, hydrogen peroxide, transfection. Hücreiçi Katalaz’ın Arttırılması ile .
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