Related Experiment Videos
Inhibition of CD95/Fas-induced DNA degradation by osmotic cell shrinkage
A C Uhlemann1, C Müller, J Madlung
1Dept. of Physiology I, University of Tübingen, Gmelinstrasse 5, 72076 Tübingen, Germany.
Abstract:
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.
Insights
Osmotic cell shrinkage inhibits CD95-induced DNA fragmentation and DNase activation in lymphocytes. This suggests distinct pathways for programmed cell death signaling, with DNase activation being sensitive to osmotic stress.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is vital for immune system development and function.
- Lymphocyte apoptosis is critical for immune homeostasis.
- Previous studies indicated moderate osmotic cell shrinkage interferes with CD95-induced cell death.
Purpose of the Study:
- To elucidate the mechanisms by which osmotic cell shrinkage affects CD95-receptor-mediated apoptosis in Jurkat T-lymphocytes.
- To investigate the impact of osmotic shrinkage on specific apoptotic signaling pathways.
Main Methods:
- Jurkat T-lymphocytes were treated with anti-CD95 antibody to induce apoptosis.
- Osmotic cell shrinkage was induced using 100 mM NaCl.
- Apoptosis markers including phosphatidylserine exposure, caspase 3 activity, DNA fragmentation, and DNase activation were assessed.
- Western blot analysis was used to examine protein phosphorylation and kinase activation (p56lck).
Main Results:
- Osmotic shrinkage did not significantly affect CD95-induced phosphatidylserine exposure or caspase 3 activation.
- However, osmotic shrinkage markedly inhibited CD95-induced DNA fragmentation and DNase activation.
- CD95-induced tyrosine phosphorylation of a ~20 kD nuclear protein and p56lck activation were blunted by osmotic shrinkage.
Conclusions:
- Distinct signaling pathways mediate different aspects of CD95-induced apoptosis.
- DNase activation, a key event in DNA fragmentation, is particularly sensitive to osmotic cell shrinkage.
- These findings highlight the role of osmotic stress in regulating programmed cell death pathways in lymphocytes.