Caspase-3 activates endo-exonuclease: further evidence for a role of the nuclease in apoptosis.
Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130-185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 microM etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells.
Department of Immunology and Rheumatology, Sydney Children's Hospital, Randwick, NSW, Australia.
X W
XW
Meng
M J
MJ
Fraser
J M
JM
Feller
J B
JB
Ziegler
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology; D002843:Chromatin / Q000378:metabolism; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D005092:Exonucleases / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting
D002843:Chromatin; D005047:Etoposide; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases