Authors: H. BARBAROS ORAL, ANDREW J.T. GEORGE, DORIAN O. HASKARD
Abstract: Apoptosis induced by hydrogen peroxide (H_2O_2 ) 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 H_2O_2 (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 H_2O_2 -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 H_2O_2 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 H_2O_2 -dependent pathway.
Keywords: Apoptosis, catalase, cisplatin, hydrogen peroxide, transfection.
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