Abstract
Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization 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, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic 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 immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.
Highlights
Apoptosis is a physiological process of orderly cell death that occurs in response to a number of physiological, pathological, and cytotoxic insults
A 70-kDa Endonuclease Is Exclusively Activated in Hematopoietic Cells—To identify the size and activity of putative apoptotic endonucleases, we fractionated whole cell lysates from apoptotic cells on DNA-SDS-PAGE gels under denaturing conditions using a technique adapted from McGrew and Green [15]
We have identified and purified a 70-kDa endonuclease (NUC70) the expression of which is confined to cells of hematopoietic lineage
Summary
Reagents and Materials—Vincristine, cycloheximide, actinomycin D, VP-16, and chemicals and protease inhibitors for buffers were obtained from Sigma. The nuclei pellet was washed twice with the above buffer, and nuclear protein was extracted with 0.3 M NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM PMSF, 2 mM EDTA, 0.5 g/ml leupeptin at 4 °C for 1 h on a moving platform. Protein concentrations were determined by the method of Bradford [14] using bovine serum albumin as standard Both cytoplasmic and nuclear extracts were prepared from cells during apoptosis at different time points and analyzed using the DNASDS-PAGE method described below. Fractions with endonuclease activity were pooled and reconcentrated in equilibration buffer with 50 mM NaCl. The sample was loaded onto a double-stranded DNA-agarose column (Amersham Pharmacia Biotech) and eluted in a manner similar to that described above. Figures were scanned using Microtek ScanMaker IIXE and assembled using Adobe Photoshop
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