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DT40 cells lacking the Ca2+-binding protein annexin 5 are resistant to Ca2+-dependent apoptosis
Timothy E Hawkins1, Debipriya Das, Barry Young
1Department of Physiology and Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Abstract:
Annexins are widely expressed Ca(2+)-dependent phospholipid-binding proteins with poorly understood physiological roles. Proposed functions include Ca(2+) channel activity and vesicle trafficking, but neither have been proven in vivo. Here we used targeted gene disruption to generate B-lymphocytes lacking annexin 5 (Anx5) expression and show that this results in reduced susceptibility to a range of apoptotic stimuli. By comparison B-lymphocytes lacking annexin 2 (Anx2) showed no such resistance, providing evidence that this effect is specific to loss of Anx5. The defect in the ANX5(-/-) cells occurs early in the apoptotic program before nuclear condensation, caspase 3 activation, and cell shrinkage, but downstream of an initial Ca(2+) influx. Only UVA/B irradiation induced similar levels of apoptosis in wild-type and ANX5(-/-) cells. Unexpectedly, ANX5(-/-) cells permeabilized in vitro also failed to release mitochondrial cytochrome C, suggesting a possible mechanism for their resistance to apoptosis. These findings demonstrate a role for Anx5 in determining the susceptibility of B-lymphocytes to apoptosis.
Insights
Loss of annexin 5 (Anx5) in B-lymphocytes reduces apoptosis susceptibility. ANX5(-/-) cells resist cell death by preventing mitochondrial cytochrome C release, highlighting Anx5
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Annexins are calcium-dependent phospholipid-binding proteins with unclear physiological functions.
- Proposed roles in calcium channel activity and vesicle transport lack in vivo validation.
Purpose of the Study:
- To investigate the in vivo role of annexin 5 (Anx5) in B-lymphocyte apoptosis.
- To determine if Anx5 deficiency impacts susceptibility to apoptotic stimuli.
Main Methods:
- Targeted gene disruption to create Anx5-deficient (ANX5(-/-)) B-lymphocytes.
- Comparison of apoptosis induction in ANX5(-/-) and Anx2-deficient B-lymphocytes versus wild-type cells.
- Analysis of apoptotic markers including nuclear condensation, caspase 3 activation, and cytochrome C release.
Main Results:
- ANX5(-/-) B-lymphocytes exhibited reduced susceptibility to various apoptotic stimuli, unlike Anx2-deficient cells.
- The defect in ANX5(-/-) cells occurred early in apoptosis, preceding caspase activation but following calcium influx.
- ANX5(-/-) cells failed to release mitochondrial cytochrome C upon in vitro permeabilization, suggesting a resistance mechanism.
- UVA/B irradiation was the only stimulus that induced comparable apoptosis levels in wild-type and ANX5(-/-) cells.
Conclusions:
- Annexin 5 plays a significant role in determining B-lymphocyte susceptibility to apoptosis.
- The absence of Anx5 confers resistance to apoptosis, potentially via impaired mitochondrial cytochrome C release.
- These findings provide crucial in vivo evidence for Anx5 function in regulating programmed cell death in lymphocytes.