Effector caspases are dispensable for the early nuclear morphological changes during chemical-induced apoptosis.
Nuclear morphological changes during apoptosis are very distinct and effector caspases have been implicated to play a central role in these processes. To investigate this in greater detail we examined the effect of blocking caspase activity and its activation on the nuclear morphological change in Jurkat T cells undergoing apoptosis after staurosporine treatment. In the presence of caspase inhibitors, like benzyloxycarbonyl-Val-Ala-Asp fluoro-methylketone (z-VAD-FMK), N-acetyl Tyr-Val-Ala-Asp chloromethylketone (Ac-YVAD-CMK) and benzyloxy-carbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-FMK), staurosporine-treated Jurkat cells displayed a nuclear morphological change distinct from that of normal and apoptotic cells. This nuclear morphological change is an early event, characterised by convoluted nuclei with cavitations, and clumps of chromatin abutting to inner regions of the nuclear envelope between the nuclear pores. Both the nuclear envelope and endoplasmic reticulum were grossly dilated. This pre-apoptotic nuclear change precedes the externalisation of phosphatidylserine, chromatin condensation and DNA laddering, and can be dissociated from the formation of high molecular weight DNA fragments and cell shrinkage. Although cytochrome c efflux from the mitochondria and the processing of caspase-3 were observed in Jurkat cells with pre-apoptotic nuclear morphology, caspase-2, -6, -7 and -8 were not activated. In the presence of z-DEVD-FMK or Ac-YVAD-CMK, caspase-3 was processed to both the p17 and p20 fragments in staurosporine-treated cells, but only to p20 fragment in the presence of z-VAD-FMK. However, the caspase-3 substrate, poly(ADP ribose) polymerase was not cleaved in the presence of z-VAD-FMK, despite >70% of the cells have pre-apoptotic nuclei. In addition, caspase-3 null MCF-7 cells also undergo pre-apoptotic nuclear change when treated with staurosporine in the presence of caspase inhibitors, indicating that caspase-3 is not required for the early nuclear morphological change in cells undergoing apoptosis. Although cell death in staurosporine-treated Jurkat cells was markedly delayed, they eventually die without discernible downstream apoptotic features. Other apoptotic stimuli like etoposide and the heavy metal chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine also induced this nuclear morphological change in Jurkat cells in the presence of z-VAD-FMK. In summary, the effector caspases are not involved in early nuclear morphological change, which precedes the conventional hallmark morphological changes associated with chemical-induced apoptosis.
Centre for Mechanisms of Human Toxicity, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK.
V L
VL
Johnson
S C
SC
Ko
T H
TH
Holmstrom
J E
JE
Eriksson
S C
SC
Chow
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D002614:Chelating Agents / Q000494:pharmacology; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation; D009685:Nuclear Envelope / Q000187:drug effects / Q000648:ultrastructure; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002614:Chelating Agents; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005029:Ethylenediamines; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D019384:Nucleic Acid Synthesis Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D019311:Staurosporine; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine