Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro.
DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.
Choi
J-J
JJ
Medical Research Service, Durham VA Medical Center, Division of Rheumatology, Duke University Medical Center, 508 Fulton Street, Durham, NC 27705, USA.
Reich
C F
CF
Pisetsky
D S
DS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5; D017209:Apoptosis / Q000235:genetics / Q000276:immunology*; D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000276:immunology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D009336:Necrosis; D009930:Organic Chemicals; D011419:Propidium; D018709:Statistics, Nonparametric; D019311:Staurosporine / Q000494:pharmacology; D020298:U937 Cells
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; D005047:Etoposide; D004247:DNA; D019311:Staurosporine; D002166:Camptothecin