The purpose of this proposal is to study the role of active oxygen species in cancer cell cytotoxicity and to determine if lipids are a major target of these species. In order to study the role of active oxygen, superoxide dismutase (SOD) levels of parental and doxorubicin- resistant human breast carcinoma MCF-7 cells will be modulated and the effect on cell killing by selected antitumor agents (doxorubicin, bleomycin, ionizing radiation, tumor necrosis factor) determined. Modulation of superoxide dismutase will be accomplished by transfection of sense and anti-sense SOD cDNA. After transfection, clones with lower and higher than control levels of SOD activity will be isolated. These clones will then be tested for sensitivity to the selected antitumor agents. Both CuZnSOD and MnSOD will be transfected. Verification that superoxide dismutase modulation has occurred will be accomplished using enzyme activity, immunoreactive protein, Northern and Southern blot assays. Catalase, glutathione peroxidase, glutathione reductase, and glutathione levels will also be measured in the stable clones to determine if any of the other common antioxidants have been altered. Similar studies will be performed after glutathione peroxidase (GPX) transfection. GPX will be transferred into wild type MCF-7 cells as well as an SOD-overexpressing clone. The role of oxygen and lipid radicals will also be tested directly by measuring qualitatively and quantitatively the radicals present after treatment of control and SOD-and/or GPX-modified cells with the selected antitumor agents. Measurement of radicals will be accompanied by using the electron paramagnetic resonance technique of spin trapping. Last, the role of lipids in the antitumor effect will be studied. This will be done in two different ways: 1) measuring the amount of lipid peroxidation that occurs during tumor cell killing by the above agents; and 2) modifying the lipids of the cell and determining the effect on tumor cell killing by selected antitumor combinations in control and SOD- and/or GPX-transfected cell lines. This research should provide valuable new information on the mechanisms and targets of antitumor agents. It is hoped this knowledge will eventually lead to more powerful antitumor protocols.
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