Cytochrome c release and endoplasmic reticulum stress are involved in caspase-dependent apoptosis induced by G418

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...

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Bibliographic Details
Published in:Cellular and molecular life sciences : CMLS 2004-07, Vol.61 (14), p.1816-1825
Main Authors: Jin, Q H, Zhao, B, Zhang, X J
Format: Article
Language:English
Subjects:
Animals
Anti-Bacterial Agents - pharmacology
Apoptosis
Apoptosis - drug effects
Caspases - drug effects
Cells
Cytochromes c - drug effects
Endoplasmic Reticulum - drug effects
Gentamicins - pharmacology
Kidney - cytology
Kidney - drug effects
Kidney - ultrastructure
Microscopy, Electron
Microscopy, Fluorescence
Proteins
Rats
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Summary: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 indicated that these two pathways were independent of each other.
ISSN:1420-682X
1420-9071
DOI:10.1007/s00018-004-4143-7