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First published online 15 January 2008
doi: 10.1242/jcs.017343

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Cytoplasmic condensation is both necessary and sufficient to induce apoptotic cell death

Department of Neurobiology, the Civitan International Research Center, and the Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (36K): [in this window] [in a new window]   Fig. 1. Apoptosis is initiated in human glioma cells in response to staurosporine (STS) and TRAIL treatment. (A) Caspase activity was measured in D54-MG cells treated with normal bath, 1 µM STS or 50 ng/ml TRAIL by a luminescence assay. Each time point was normalized to the luminescence of cells after 0 hours of treatment with normal bath. Data are from three experiments. Error bars represent s.e.m. and n=3 samples per experiment. (B) Western blots of cell lysates from cells treated for 1, 3 or 7 hours were probed with antibodies against the cleaved, activated forms of caspases 3, 8 and 9. (C) Representative images demonstrate DNA fragmentation (fluorescein-labeled nuclei) after treatment with normal bath, STS or TRAIL for 7 hours. Merged images show both fragmented nuclei (green) and total nuclei present by DAPI staining (blue). Bar, 100 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Apoptotic cell condensation is sensitive to the Cl–-channel inhibitor DIDS. (A,B) Normalized mean cell volumes (MCVs) of D54-MG were recorded following addition of STS (A) or TRAIL (B) in the presence of the Cl–-channel inhibitors NPPB, DIDS or Cd2+ or the KCC inhibitor DIOA. Data are the mean results from three independent experiments. Error bars represent s.e.m. with n=10,000-20,000 cells.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Apoptosis initiation results in DIDS-sensitive Cl– movement across the plasma membrane. (A) Representative images depict cell size as well as SPQ and GFP intensity just prior to (t=0) and 27 minutes after the addition of control or STS media. (B) Mean time normalized intensity curves quantify the changes depicted in A. Data are the mean results from three independent experiments. Error bars represent s.e.m. with n=10 samples in each experiment.

View larger version (31K): [in this window] [in a new window]   Fig. 4. DNA fragmentation and caspase activation are also sensitive to DIDS inhibition of AVD. (A) Representative images demonstrate the presence or absence of DNA fragmentation (FITC-labeled nuclei) in cells treated for 7 hours with STS or TRAIL in the presence or absence of DIDS. Merged images show fragmented nuclei (green) as well as DAPI staining of all nuclei present (blue). (B) Western blots of lysates from similarly treated cells were probed with antibodies to activated, cleaved caspases 3, 8 and 9. Actin was used as a loading control.

View larger version (35K): [in this window] [in a new window]   Fig. 5. DIDS inhibition of AVD has variable effects on cell viability. Percentage cell viability as measured by Trypan Blue exclusion after application of STS or TRAIL for 7 hours. *P<0.05 compared with untreated control cells.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Hyperosmotic cell shrinkage results in activation of caspase 3. (A) Normalized MCVs of D54-MG cells were recorded following the addition of 150 or 300 mOsm mannitol in the presence or absence of 2 mM glutamine. Data are the mean results from three independent experiments. Error bars represent s.e.m. with n=10,000-20,000 cells. (B) Caspase 3 activity, as measured by luminescence, in cells exposed to isosmotic (300 mOsm) or hyperosmotic (337.5-600 mOsm) bath solutions for 24 hours. Activity normalized to 300 mOsm exposure. Data are from three experiments. Error bars represent s.e.m. and n=3 samples per experiment. *P<0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 7. The `time threshold' to activate is reduced with a greater cell shrinkage. Cells were exposed to bath solution made hyperosmotic by the addition of NaCl (final osmolarity 450 or 600) and were then `rescued' from shrinkage by the addition of ddH2O after 30 minutes, 1, 4 or 8 hours. (A) Normalized MCVs of D54-MG cells during exposure to and rescue from hypertonic challenge in a representative experiment. (B) Caspase 3 activity as measured by luminescence. Activity is normalized to 300 mOsm (no challenge) exposure. Data are from three independent experiments. Error bars represent s.e.m. and n=3 samples per experiment. *P<0.05 compared to unchallenged control cells (not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Proposed model for the initiation of apoptosis by cell shrinkage. Cell shrinkage may result in the increased concentration of auto-activating enzymes (top panel) or the increase in proximity of cell surface receptors (bottom panel). The activation of such enzymes/receptors may result in the initiation of apoptosis in the absence of other drugs or the acceleration of the pathway in the presence of other apoptosis initiators.