We previously developed a new microscopic observation system that enables time-lapse quantitative analysis of apoptosis and necrosis. With this system we quantitatively analyzed adriamycin (ADR)-induced cell death using manganese superoxide dismutase (MnSOD)- and wild-type p53-gene transfectants on SaOS2, a p53-deficient human osteosarcoma cell line. A highly MnSOD-overexpressing cell line, SaOS2FM(H), acquired ADR-tolerance compared to the parent cell line SaOS2. The ADR-tolerance of SaOS2FM(H) diminished by L-buthionine-[S,R]-sulfoximine (BSO), which did not change ADR-sensitivity of SaOS2, to the similar ADR-sensitivity of SaOS2. A wild-type p53-expressing cell line, SaOS2wtp53, significantly increased in ADR-sensitivity compared to SaOS2. This ADR-sensitivity of SaOS2wtp53 was enhanced by BSO. When isosorbide 5-mononitrate was combined with BSO, isosorbide 5-mononitrate increased ADR sensitivity of a moderately MnSOD-overexpressing cell line, SaOS2FM(L), decreased that of SaOS2 FM(H), and did not change those of SaOS2 and SaOS2wtp53 compared to BSO alone. Time-lapse microscopic observations during ADR treatment for 24 h indicated that the most cells of each cell line underwent apoptosis, and a few cells (less than 11%) died by necrosis. When cells were treated with iso-concentration of ADR, apoptosis of SaOS2FM(H) was less than that of SaOS2. BSO, which did not change ADR-sensitivity of SaOS2, increased appearance rate of ADR-induced apoptosis, but not necrosis of MnSOD-overexpressing cell lines. When iso-survival dose of ADR, which reduced surviving fraction to 0.01, was given for each cell line, no difference was observed in appearance of either apoptosis or necrosis between SaOS2 and MnSOD-overexpressing cell lines. On the other hands, appearance of both apoptosis and the following secondary necrosis of SaOS2 wtp53 was significantly accelerated compared to those of SaOS2. These findings indicate that hydrogen peroxide overload on p53-independent pathway due to MnSOD overexpression plus BSO might increase the apoptosis frequency without acceleration of apoptotic process of each cell, resulting in negating ADR-tolerance of MnSOD-overexpressing cell lines.

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