Confocal Microscopic Analysis of the Interaction between Cisplatin and the Copper Transporter ATP7B in Human Ovarian Carcinoma Cells

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Abstract
Some cisplatin (DDP)-resistant cells overexpress the copper export transporter ATP7B, and cells molecularly engineered to overexpress ATP7B are resistant to DDP. The interaction of Cu with ATP7B normally triggers its relocalization from the perinuclear region to more peripheral vesicles. To investigate the interaction of DDP with ATP7B, we examined the effect of DDP on the subcellular localization of ATP7B using human ovarian carcinoma cells expressing a cyan fluorescent protein (ECFP)-tagged ATP7B (2008/ECFP-ATP7B). ATP7B expression was confirmed in 2008/ECFP-ATP7B cells by Western blotting, and its functionality was documented by showing that it rendered the cells 1.9-fold resistant to CuSO4 and 4.1-fold resistant to DDP and also reduced the accumulation of both drugs. There was greater sequestration of Pt into intracellular vesicles in the 2008/ECFP-ATP7B cells than in the 2008/ECFP cells. Confocal digital microscopy revealed that ECFP-ATP7B localized in the perinuclear region in absence of drug exposure and that both Cu and DDP triggered relocalization to more peripheral vesicular structures. A fluorescein-labeled form of DDP that retained cytotoxicity and was subject to the same mechanisms of resistance as DDP colocalized with ECFP-ATP7B in the 2008/ECFP-ATP7B cells, whereas the same fluorochrome lacking the DDP moiety did not. These results provide evidence that DDP directly interacts with ATP7B to trigger its relocalization and that ATP7B mediates resistance to DDP by sequestering it into vesicles of the secretory pathway for export from the cell.
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