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.

We show that H2O2 increases acetylcholinesterase (AChE) expression via transcriptional activation through c-Jun N-terminal kinase (JNK), since the JNK inhibitor SP600125, but not the extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059 or p38 kinase inhibitor SB203580, attenuated H2O2-induced AChE expression and its promoter activity. Overexpression of hemagglutinin (HA)-JNK increases H2O2-induced AChE expression and its promoter activity, whereas the dominant negative mutant form of JNK suppressed H2O2-induced AChE expression and promoter activity. Mutation analysis indicates that the major response elements for JNK in the AChE promoter are the AP1-like element (TGAGTCT) site, located within the -1565/-1569 region of the AChE promoter, and the ATF2 element (CCACGTCA), within the -2185/-2177 region. The AP1-like element binds to the transcription factors, c-jun and ATF2, while the ATF2 element binds mainly ATF2. Taken together, our results strongly suggest that H2O2 induces AChE expression via the JNK/AP1/ ATF2 signaling pathway.