Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.
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Acknowledgements. We thank Drs. T Yoshimori and N Mizushima for sharing the GFP-LC3 plasmid, Drs. Doug Yee, Carol Lange and Vitaly Polunovsky for sharing breast cell lines, Dr. Robert Hafner for help with the transmission electron microscopy, and Jerry Sedgewick and John Oja for help with the confocal microscopy. We also thank Dr. Thomas Neufeld for helpful discussions. This work was supported by research grants from the Minnesota Medical Foundation and from The University of Minnesota Cancer Center, a Comprehensive Cancer Center designated by the National Cancer Institute and the Regis Foundation.