There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1β– converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode “death” gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t -butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage–induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing. Footnotes Part of this work was supported by an MRC-DTI LINK award to G.I. Evan and by a Concerted Action grant (No. BMH1-C794-1471) to G.I. Evan and N.J. McCarthy. M. Whyte was supported by an Advanced Clinical Training Fellowship from the Wellcome Trust (Fellowship No. 041759). We are also indebted to the students of Bishop Stopford School for their generous donations in memory of Elliot Smith, Simon Railton, Joanne Briellat, and to whom this work is dedicated. Note Added in Proof. Quick-Time movies showing c-Myc–induced apoptosis in the absence of serum (Movie One) and c-Myc–induced apoptosis in the absence of serum and the presence of 100 μM zVAD.fmk, the blebbing phenotype (Movie Two), are available from the following Web site address: http://www.icnet.uk/axp/bcn/ Address all correspondence to Gerard Evan, Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Tel.: (44) 171 269 3439. Fax: (44) 171 269 3230. E-mail G.Evan@icrf.icnet.uk Abbreviations used in this paper: 4-OHT 4-hydroxytamoxifen BD.fmk t- butoxy carbonyl-Asp.fluoromethylketone FLICE FADD-like ICE ICE interleukin-1β-converting enzyme IGF insulin-like growth factor IRP ICE-related protease MACH MORT1-associated CED-3 homologue MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PARP poly-(ADP-ribose) polymerase RT room temperature ZVAD.fmk benzyloxycarbonyl-Val-Ala-Asp( O- methyl)-fluoromethylketone Submitted: 11 July 1996 Revision received 22 October 1996
The Journal of Cell Biology – Rockefeller University Press
Published: Jan 13, 1997
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