Inhibition of CD95/Fas-induced DNA degradation by osmotic cell shrinkage.
Apoptosis (programmed cell death) is an active physiological mechanism from which removal of abundant or potentially harmful cells follows. Apoptosis of lymphocytes is critical for the development of the immune system and during the immune response. As we have shown previously, moderate osmotic cell shrinkage interferes with CD95(Fas/Apo-1)-induced cell death. The present study has been performed to further elucidate the underlying mechanisms. To this end, apoptosis in Jurkat T-lymphocytes was elicited by triggering the CD95-receptor with monoclonal CD95/Fas-antibody. Osmotic cell shrinkage which was induced by the addition of 100 mM NaCl, did not significantly interfere with CD95-induced phosphatidylserine exposure nor the activation of caspase 3 activity as determined by PARP cleavage, DEVD-AMC consumption, or the activation of PAK2-kinase. However, osmotic cell shrinkage almost abolished CD95-induced DNA fragmentation (as revealed by propidium iodide staining) and the activation of a DNase as evidenced from SDS-PAGE gel assay. Western blot analysis showed CD95-induced tyrosine phosphorylation of a nuclear protein of ca. 20 kD which comigrated with nuclease activity. This tyrosine phosphorylation was almost completely abolished by the addition of 100 mM NaCl. Furthermore, osmotic cell shrinkage blunted the CD95-induced activation of the Src-like kinase p56lck. It is concluded that different signaling pathways mediate FITC-Annexin-V binding and DNase activation. Only the latter is sensitive to osmotic cell shrinkage.
Uhlemann
A C
AC
Dept. of Physiology I, University of Tübingen, Gmelinstrasse 5, 72076 Tübingen, Germany.
Müller
C
C
Madlung
J
J
Gulbins
E
E
Lang
F
F
D017304:Annexin A5 / Q000378:metabolism; D001692:Biological Transport; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism*
D017304:Annexin A5; D010718:Phosphatidylserines; D019014:fas Receptor; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D003851:Deoxyribonucleases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases