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

Cellular and Molecular Life Sciences : CMLS
Q H JinX J Zhang

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

Apr 5, 2013·Seminars in Immunopathology·Masanori Kitamura
Oct 31, 2007·Apoptosis : an International Journal on Programmed Cell Death·H ServaisM-P Mingeot-Leclercq
Feb 17, 2010·Cell Research·Qihuang JinQiwei Zhai
Jun 11, 2009·Antioxidants & Redox Signaling·Jeffrey G Dickhout, Joan C Krepinsky
Nov 1, 2005·Drug Metabolism Reviews·Alastair E CribbLaetitia Schneider
Jun 3, 2010·Experimental Parasitology·Virginia Sánchez MonroyDavid Guillermo Pérez Ishiwara
Aug 30, 2008·Mitochondrion·Young Geol Yoon, Michael D Koob
Jul 24, 2010·British Journal of Pharmacology·Jian YaoMasanori Kitamura
May 23, 2012·Therapeutic Apheresis and Dialysis : Official Peer-reviewed Journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy·Jianhui ZhouXiangmei Chen
May 23, 2007·Journal of Cellular Physiology·Qihuang JinQiwei Zhai
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Mar 28, 2008·American Journal of Physiology. Renal Physiology·Masanori Kitamura
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Jan 29, 2016·Chembiochem : a European Journal of Chemical Biology·Gutmann MarcusTessa Lühmann
Jun 10, 2008·Clinical and Experimental Nephrology·Masanori Kitamura

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