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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02580

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Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway

Subbareddy Maddika^1,2,*, Evan P. Booy^1,2,*, Dina Johar^1,2, Spencer B. Gibson^1,2, Saeid Ghavami^1,2 and Marek Los^1,2,3,

¹ Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, MB R3E OV9, Canada
² Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB R3E OV9, Canada
³ The Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4, Canada

View larger version (37K): [in a new window]   Fig. 1. Apoptin-induced cell death is independent of FADD and caspase-8. (A) MCF7 and PC-3 cells treated with recombinant TAT-GFP (1 µM) or with recombinant TAT-apoptin (1 µM), and counterstained with DAPI. TAT-GFP localizes both to the nucleus and to the cytoplasm, whereas TAT-apoptin is found almost exclusively in the nuclei (arrows). TAT-apoptin was detected using an anti-haemagglutinin-tag antibody; the tag is incorporated between TAT and the apoptin itself within the fusion protein. Cells treated with TAT-GFP show normal morphology, whereas some of the cells treated with TAT-apoptin show typical apoptotic morphology (cell shrinkage, nuclear condensation). (B) Apoptosis detection in peripheral blood mononuclear cells treated with recombinant TAT-GFP (1 µM) or recombinant TAT-apoptin (1 µM) for the indicated time, or with 5 µg/ml Actinomycin D as a positive control. Some samples were also treated with IL-2 (5 U) in order to counteract the spontaneous apoptosis. (C,D) Measurement of apoptosis in Jurkat caspase-8(-/-) cells (C), Jurkat cells expressing FADD-DN (D), and control cells treated with TAT-apoptin for the indicated time periods. To exclude solvent- or TAT-peptide related effects, some control cells were treated with the recombinant TAT-GFP (1 µM) instead of TAT-apoptin treatment. (E) Apoptosis detection, measured by flow cytometry, in Jurkat cells either left untreated or treated with the agonistic anti-CD95 antibody (0.1 µg/ml) for 8 hours. Treated Jurkat caspase-8(-/-) cells and Jurkat cells expressing FADD-DN are also shown. The percentages of apoptotic cells (mean values of three independent experiments) are shown. (F) Jurkat and peripheral blood mononuclear cells (PBLs), isolated by Ficoll-PaqueTM PLUS gradient (Amersham Biosciences) treated with the recombinant TAT-Apoptin (1 µM) for 12 hours. TAT-apoptin was detected by an anti-HA antibody, as in A. Cells are counterstained with DAPI.

View larger version (15K): [in a new window]   Fig. 2. Blocking of CD95/Fas has no effect on apoptin-induced cell death. (A) Flow cytometry analysis of apoptosis in Jurkat cells, either grown in control medium or incubated with a human anti-APO-1-IgG1 (a neutralizing antibody that blocks the interaction of CD95/APO-1/Fas and the ligand), treated with TAT-apoptin for the indicated time points. To maintain the blockage, treatment with the blocking antibody was repeated every 8 hours. To exclude solvent- or TAT-peptide-related effects, some control cells were treated with recombinant TAT-GFP (1 µM) instead of TAT-apoptin. (B) Control experiment to assess cell death mediated by CD95. Both control cells and the Fas-neutralized cells were treated with an anti-Fas (IgM) antibody, and cell death was measured after 8 hours.

View larger version (18K): [in a new window]   Fig. 3. Mitochondrial membrane potential is lost during apoptin treatment, and anti-apoptotic Bcl-2 family members block apoptin-induced death. (A) Mitochondrial membrane potential (m) determination in Jurkat control cells, caspase-8(-/-) Jurkat cells and FADD-DN Jurkat cells either left untreated or treated with TAT-apoptin for the indicated time periods. m was determined by using a cationic carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), which shows a decrease in the red fluorescence upon loss of membrane potential. (B) Apoptosis (hypodiploid nuclei) detection, measured by flow cytometry, in Jurkat cells and clones stably transfected with either Bcl-2 or Bcl-XL treated with TAT-apoptin (1 µM) for the indicated time. (C) MCF7, MCF7-Bcl-2 and MCF7-caspase-3 breast cancer adenocarcinoma cells treated with TAT-Apoptin (1 µM) for the indicated period of time assessed for cell viability by the MTT assay. To exclude solvent- or TAT-peptide-related effects, some control cells (A-C) were treated with recombinant TAT-GFP (1 µM) instead of TAT-apoptin.

View larger version (68K): [in a new window]   Fig. 4. Apoptin triggers the release of pro-apoptotic mitochondrial proteins. (A) Western blot analysis of cytochrome c and AIF in mitochondrial (Mitoch.), cytosolic (Cytosol) and nuclear fractions of Jurkat cells treated with apoptin. (B) The precise localization of AIF in cells, as determined by confocal microscopy. MCF7 cells were either left untreated or stimulated with TAT-apoptin (1 µM). After 36 hours, cells were immunostained with an anti-AIF antibody followed by the corresponding Cy-3 conjugated secondary antibody (red) and with an anti-HA-antibody (TAT-apoptin has an incorporated HA-tag) followed by the detection with a FITC-labelled antibody (green). Cells were counterstained with DAPI to visualize nuclei.

View larger version (46K): [in a new window]   Fig. 5. Apoptin activates caspase-3 but not caspase-8. Western blot analysis of cell lysates from Jurkat cells treated with apoptin in the presence or absence of a caspase inhibitor zVAD-fmk (40 µM) for 0, 48 and 60 hours to detect the levels of caspase-3 and caspase-8, and cleavage. Jurkat cells treated with TAT-GFP are shown as a control. The molecular sizes of pro-caspase molecules and the active fragments are indicated.

View larger version (55K): [in a new window]   Fig. 6. Apoptin-induced cell death depends on Nur77, and involves its nucleo-cytoplasmic translocation. (A) Detection of Nur77 in MCF7 cells lipofected with scrambled or Nur77-targeting siRNA; protein was extracted at 0, 12, 24 and 30 hours post transfection. Tubulin was included as a loading control. (B) Cell viability of MCF7 cells treated with TAT-GFP (Control), transfected with Nur77 siRNA, or treated with TAT-apoptin (1 µM) either alone or in the presence of Nur77 siRNA, as determined by MTT assay at the indicated time points. (C) Confocal microscopy visualization of Nur77 (green) in MCF7 cells either left untreated or treated with TAT-apoptin for 30 hours. Mitochondria are stained by Mitotracker® (red), nuclei with DAPI (blue).

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