Studying the Effects of Various Agents on Mammalian Cell Mitochondria Using the Fluorescent Dye, Rhodamine-123
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Rhodamine-123 (Rh123) is a cationic fluorescent dye that has been shown to stain selectively the mitochondria of living cells. In this study, tumorigenic and normal mammalian cell lines were stained with Rh123. First, appropriate staining conditions were established, which were non-toxic to the cells. While the tumorigenic cell growth was inhibited by higher Rh123 concentrations at longer exposure times, normal cell growth was hot affected. Second, two tumorigenic cell lines were exposed to various mitochondrial damaging agents, namely electron transport inhibitors and membrane ionophores. Following this treatment, the Rh123 staining of these cells was quantitated using a fluorescence microscope or a Fluorescence Activated Cell Sorter. In most cases, short term (approximately 2hr.) exposure to these agents caused a marked reduction in (non-toxic) RHl23.staining, indicating mitochondrial damage. Finally, Rh123 staining was used to determine the effects of the antitumor agent CC-1065on tumorigenic cells. CC-1065 is an Upjohn drug believed to bind to mitochondrial DNA and halt replication. However, no difference in control cell vs treated cell staining was observed until after 1-2 days of continuous exposure. Electron microscope studies also showed that CC-1065 exposure did not significantly damage the mitochondria of B-16 cells. The lack of staining and morphological changes in the mitochondria, in spite of the observation of CC-1065's cytotoxicity in growth assays, suggest that the cytotoxic effects of CC-1065 are due to the effects of the drug at some other site.