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Anthracyclines are effective antitumor agents whose chief limitation has been cardiotoxicity directly related to free radical production. Therefore, strategies designed to selectively overexpress antioxidant proteins in the heart could protect against drug-induced toxicity and allow higher doses of chemotherapy. However, to date an adequate cardiac model system that is susceptible to anthracycline injury and can express foreign genes in a controlled fashion has been lacking. Developing a cardiac model system would permit examination of the relationship between the expression level of a potentially protective foreign gene and the degree of protection from injury. In this study we have examined the potential of the H9C2 rat cardiac myocyte cell line in this regard. H9C2 cells differentiate in a reproducible fashion, as shown by progressive increases in muscle tropomyosin-expressing cells, the organization of this thin filament protein, and the percentage of muscle cells contained within myotubes. Exposure of this cell line to the anthracycline doxorubicin produces cell injury as indicated by release of the intracellular enzyme lactate dehydrogenase into the culture medium. This injury is preceded by generation of reactive oxygen species, indicated by fluorescence after loading with carboxy-dichlorodihydrofluorescein diacetate. Stable transfection of H9C2 cells with a plasmid producing a tetracycline transactivator protein allows foreign genes to be expressed at a level tightly controlled by the concentration of tetracycline in the culture medium. Since H9C2 cells differentiate, can be injured by anthracycline exposure, and can express foreign genes at controllable levels, this is a suitable system in which to design genetic approaches to prevent this important clinical problem., (Copyright 2001 Academic Press.)