A. Urbano et al., Isolation and characterization of NUC70, a cytoplasmic, hematopoietic apoptotic endonuclease, J BIOL CHEM, 273(52), 1998, pp. 34820-34827
Endonucleolytic DNA fragmentation is the common end point and the prevailin
g indicator of apoptosis, We have identified a 70-kDa endonuclease (NUC70)
that is activated in drug-induced apoptosis of human hematopoietic cells. W
e purified NUC7O to homogeneity and generated a rabbit polyclonal antibody
to distinguish it from previously identified nucleases. Biochemical charact
erization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and
active over a pH range of 6-8, When incubated with isolated HeLa nuclei, N
UC70 was capable of generating internucleosomal DNA fragmentation. This end
onucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-eth
ylnaleimide, spermine, and iodoacetamide. Western immunoblots using the ant
i-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indi
cate that NUC70 expression and activity is restricted to human hematopoieti
c cells. No such activity was detected in human epithelial cell lines or mu
rine hematopoietic cells. We also observed no difference in levels of NUC70
expression between apoptotic and nonapoptotic cells, suggesting that activ
ation of NUC70 may be by posttranslational modification. We demonstrate tha
t NUC70 activity is diminished in cells pretreated with the caspase inhibit
ors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytopl
asmic and nuclear endonuclease activities during apoptosis show that NUC70
is a cytoplasmic endonuclease that is translocated to the nucleus after the
initiation of apoptosis, We confirmed this with immuno staining studies us
ing anti-NUC70 antibody. These results demonstrate that NUC70 is an endogen
ous cytoplasmic endonuclease that is activated during apoptosis in a caspas
e-dependent mechanism.