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First published online 1 June 2004
doi: 10.1242/jcs.01121

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Baculovirus P35 inhibits NO-induced apoptosis in activated macrophages by inhibiting cytochrome c release

¹ Department of Pathology and Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA
² School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

View larger version (29K): [in a new window]   Fig. 1. Stable expression of P35 by transfected RAW 264.7 mouse macrophage cells as detected by RT-PCR of total RNA (A). Lane 1, clone pCMV-FLAG (vector control); lane 2, clone pCMV-FLAG/p35#3; lane 3, pCMV-FLAG/p35#7; lane 4, clone pCMV-FLAG/p35#9. (B) Immunoblot analysis of FLAG-P35 expression. Extracts (50 µg) from stably transfected vector or P35 RAW 264.7 cells were immunoblotted with anti-FLAG M2 antibodies to detect the expression of P35. Lane 1, clone pCMV-FLAG (vector); lane 2, clone pCMV-FLAG/p35#3; lane 3, pCMV-FLAG/p35#7; lane 4, clone pCMV-FLAG/p35#9.

View larger version (57K): [in a new window]   Fig. 2. P35 expression protects activated RAW 264.7 cells from undergoing NO-mediated oligonucleosomal DNA fragmentation. RAW 264.7 cells or vector- or p35-transfected clones (1x106 cells/well) in 6-well tissue culture plates were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml) or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of L-NMMA (1 mM) as indicated. Cells were also treated directly with NO donor, SNP (1 mM). After 36 hours of treatment, cells were assayed for DNA fragmentation using a radioisotope release assay (A) and agarose gel electrophoresis (B) as described in Materials and Methods. Data shown are mean±s.e.m. and are the representative of three independent experiments done in triplicates. *P<0.05 versus values of control cultures. **P<0.05 versus values of cisplatin and IFN-/LPS and IFN--treated cultures. #P<0.05 versus values for cisplatin + IFN-/LPS + IFN- or SNP treated RAW 264.7/pCMV-FLAG cells.

View larger version (39K): [in a new window]   Fig. 3. P35 expression in RAW 264.7 cells does not inhibit iNOS expression and nitrite production. RAW 264.7 cells or vector- or p35-transfected clone (1x106 cells/well) in 6-well tissue culture plates were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml), or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of L-NMMA (1 mM) as before. Cells were also treated directly with NO donor, SNP (1 mM). After 18 hours of treatment, cell extracts were examined for -iNOS expression by immunoblotting (A). Culture supernatants were assayed for nitrite after 18 hours of culture as described in Materials and Methods (B). Data shown are means±s.e.m. and are representative of three independent experiments done in triplicate. *P<0.05 versus values of control cultures. **P<0.05 versus values of cisplatin and IFN-/LPS and IFN--treated cultures.

View larger version (37K): [in a new window]   Fig. 4. P35 expression in RAW 264.7 cells inhibits apoptosis and loss of cell viability. RAW 264.7 cells or vector- or p35-transfected clones (1x106 cells/well) in 6-well tissue culture plates were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml), or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of L-NMMA (1 mM) as before. Cells were also treated directly with NO donor, SNP (1 mM). After 36 hours of treatment, cells were assayed for apoptosis (A) and relative cell viability (B). Data shown are mean±s.e.m. and are representative of three independent experiments done in triplicate. *P<0.05 versus values of control cultures. **P<0.05 versus values of cisplatin and IFN-/LPS and IFN--treated cultures. #P<0.05 versus values for cisplatin and IFN-/LPS and IFN- or SNP-treated RAW 264.7/pCMV-FLAG cells.

View larger version (27K): [in a new window]   Fig. 5. P35 inhibits mitochondrial cytochrome c release, mitochondrial depolarization and caspase activation. RAW 264.7 cells or vector- or P35-transfected clones (1x106 cells/well) in 6-well tissue culture plates were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml), or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of L-NMMA (1 mM). Cells were also treated directly with NO donor, SNP (1 mM). Cytochrome c in the cytosol and mitochondria was detected by immunoblot analysis (A). Mitochondrial depolarization was determined as described in Materials and Methods, and results were expressed as the percentage of cells with depolarized mitochondria (B). Extracts were prepared as described in Materials and Methods. Caspase-9 (C) and caspase 3 (D) activities were measured in protein extracts as described in Materials and Methods. Data shown are mean±s.e.m. and are representative of three independent experiments done in triplicate. *P<0.05 versus values of control cultures. **P<0.05 versus values of cisplatin and IFN-/LPS and IFN--treated cultures. #P<0.05 versus values for cisplatin and IFN-/LPS and IFN- or SNP-treated RAW 264.7/pCMV-FLAG cells.

View larger version (41K): [in a new window]   Fig. 6. P35 expression inhibits NO-mediated PARP cleavage in RAW 264.7 cells. RAW 264.7 cells or vector- or p35-transfected clones were treated with cisplatin (2 µg/ml) and IFN- (25 U/ml) or LPS (1 µg/ml) and IFN- (25 U/ml) in the presence or absence of LNMMA (1 mM) as before. Cells were also treated directly with NO donor, SNP (1 mM). After 36 hours of treatment, cell extracts were analyzed by immunoblotting with anti-PARP antibody to assess cleavage of PARP.

View larger version (26K): [in a new window]   Fig. 7. Model for P35-mediated inhibition of NO-induced apoptosis in activated macrophages. Mitochondrial cytochrome c release is a critical event in molecular pathway of apoptosis that triggers subsequent caspases activation and apoptotic cell death. Baculovirus protein P35 inhibits mitochondrial cytochrome c release as well as caspase activation. This also raises the possibility that activation of caspases may be the cause rather than consequence of cytochrome c release. AIF, apoptosis inducing factor; Apaf-1, apoptotic protease activating factor-1.

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