Fluorescence resonance energy transfer analysis of bid activation in living cells during ultraviolet-induced apoptosis.
Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9+/-1 h after UV irradiation, and the average duration of the activation was 75+/-10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50+/-10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.
Wu
Yinyuan
Y
Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Kim
Su-Gwan
SG
Xing
Da
D
Liu
Lei
L
Park
Joo-Cheol
JC
Chen
Tongsheng
T
Chen
Wei R
WR
D017209:Apoptosis / Q000528:radiation effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008564:Membrane Potentials / Q000528:radiation effects; D008928:Mitochondria / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone