Sensitization by 5-azacytidine toward death receptor-induced hepatic apoptosis.
5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2'-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5'-adenosyl)-L-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,alpha-tosyl-L-phenylalanine chloromethyl ketone, N,p-tosyl-L-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.
Weiland
Timo
T
Faculty of Biology, University of Konstanz, M667, D-78457 Konstanz, Germany.
Weiller
Markus
M
Künstle
Gerald
G
Wendel
Albrecht
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001374:Azacitidine / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D008113:Liver Neoplasms / Q000473:pathology / Q000601:surgery; D051379:Mice; D008856:Microscopy, Fluorescence; D053218:Receptors, Death Domain / Q000819:agonists* / Q000502:physiology*
D053218:Receptors, Death Domain; D020169:Caspases; D001374:Azacitidine