Cytochrome c release and endoplasmic reticulum stress are involved in caspase-dependent apoptosis induced by G418 |
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Authors: | Q?H?Jin B?Zhao Email author" target="_blank">X?J?ZhangEmail author |
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Institution: | (1) Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, 320 YueYang Road, 200031 Shanghai, China |
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Abstract: | G418 is used extensively in transfection experiments to select eukaryotic cells that have acquired neomycin
resistance genes, but the mechanism is still elusive. To investigate this, we treated normal rat kidney cells with
G418 for 3 days and found that the cells presented typical apoptotic features such as cell shrinkage, nuclear
fragmentation, and caspase-3 activation. However, there was no low-molecular DNA ladder. The pan caspase inhibitor
z-VAD-fmk completely inhibited this type of apoptosis, suggesting a caspase-dependent mechanism. Caspase cascades
in apoptosis induced by G418 were initiated by at least two pathways: the release of cytochrome c from
mitochondria, which was observed under confocal microscopy, and endoplasmic reticulum stress, demonstrated by the
increase in Ca2+ concentration and the cleavage of m-calpain and procaspase-12. Both
pathways activated caspase-9. Inhibition of caspase-9 activity by z-LEHD-fmk prevented most of the cells from
apoptosis, and E-64d, an inhibitor of calpain accentuated this block. The cleavage of casapse-9 and caspase-12 was
blocked only by simultaneous application of z-VAD-fmk and E-64d, but not by either alone. E-64d did not prevent
the release of cytochrome c. These results indicated that these two pathways were independent of each other. Received 1 April 2004; received after revision 21 April 2004; accepted 26 May 2004 |
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Keywords: | G418 apoptosis cytochrome c endoplasmic reticulum stress |
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