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 caspase-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 ind...Continue Reading
Citations
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