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© The Rockefeller University Press, 0021-9525/1998//225 $5.00
The Journal of Cell Biology, Volume 143, Number 1, , 1998 225-239

Transition from Caspase-dependent to Caspase-independent Mechanisms at the Onset of Apoptotic Execution

Kumiko Samejima^*, Shigenobu Toné^*, Timothy J. Kottke, Masato Enari, Hideki Sakahira, Carol A. Cooke^*, Françoise Durrieu^*, Luis M. Martins^*, Shigekazu Nagata, Scott H. Kaufmann, and William C. Earnshaw^*

^* Institute of Cell and Molecular Biology, University of Edinburgh, Kings' Buildings, Edinburgh, EH9 3JR, Scotland, United Kingdom; Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905; and Department of Genetics, Osaka University Medical School, Suita, Osaka 565, Japan

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

Key Words: apoptosis • caspases • CAD • ICAD • DFF

Abbreviations used in this paper: CAD, caspase-activated DNase; C/D extract, nonapoptotic extract from cells in the condemned phase; DAPI, 4,6-diamidino-2-phenylindole; DFF, DNA fragmentation factor; E/X extract, apoptotic extract from execution phase cells; Fr-2 and Fr-3, fractions 2 and 3 from heparin–agarose chromatography of S/M extract; ICAD, inhibitor of CAD; S/M extract, proapoptotic extract from committed phase cells; PARP, poly(ADP-ribose) polymerase.

Correspondence should be addressed to W.C. Earnshaw, Institute of Cell and Molecular Biology, University of Edinburgh, Kings' Buildings, Mayfield Road, Edinburgh, EH9 3JR, Scotland, UK. Tel.: 44-(0)131-650-7101. Fax: 44-(0)131-650-7100. E-mail: bill.earnshaw{at}ed.ac.uk

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