Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.
We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.
Lazebnik
Y A
YA
Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Cole
S
S
Cooke
C A
CA
Nelson
W G
WG
Earnshaw
W C
WC
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002474:Cell-Free System; D002645:Chickens; D002875:Chromosomes; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism; D006656:Protamine Kinase / Q000378:metabolism; D016196:S Phase; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology
D009707:Nucleosomes; D016590:Aphidicolin; D006656:Protamine Kinase; D015032:Zinc