Article Figures & Data
Figures
- FIG. 1.
VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.
- FIG. 2.
Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.
- FIG. 3.
Levels of expression of various EBV latent proteins and subcellular distributions of EBNA-LP in various BL cell lines and derived cell clones. (A) Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb, CS.1-4 anti-LMP-1 MAb, 1H4 anti-EBNA1 MAb, PE2 anti-EBNA2 MAb, or an anti-β-actin MAb (as a control for protein loading). (B and C) Total lysates (T) were fractionated into cytoplasmic (C) and nuclear (N) extracts. Equal amounts of each fraction (20 μg protein) were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. We checked that fractionation was complete by probing the membranes with MAbs recognizing a nuclear protein (topoisomerase II) and a cytoplasmic protein (IκBα). Molecular sizes are indicated to the right.
- FIG. 4.
Production levels, subcellular distributions, and effects on apoptosis of EBNA-LP after transient transfection. (A) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors and grown for 48 h. Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb or an anti-β-actin MAb (as a control for protein loading). Molecular sizes are indicated to the right. (B) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors and grown on glass coverslips; 48 h after transfection, the cells were stained with 4D3 anti-EBNA-LP MAb and GAM-Alexa 488. (C) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors; 48 h after transfection, the cells were treated with staurosporine (2.5 μM) for 6 h and stained with PI. The levels of apoptosis were assessed by determining the proportions of cells with sub-G1 DNA content by flow cytometry. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars show the standard deviations.
- FIG. 5.
Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(NeoR) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.
- FIG. 6.
PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.
- FIG. 7.
PP2A interacts with EBNA-LP in cells resistant to VT-1-induced apoptosis. Lysates obtained from various VT-1-sensitive or -resistant cells were subjected to immunoprecipitation (IP) with 4D3 anti-EBNA-LP MAb or irrelevant Abs (Irr). Western blot analysis was then performed with an anti-PP2Ac Ab or 4D3 anti-EBNA-LP MAb. As a control for protein levels before IP, a portion of cell lysate (Input) corresponding to 15% of the input for IP was also included in the Western blot analysis. Molecular sizes are indicated to the right.
Tables
- TABLE 1.
VT-1- and staurosporine-induced apoptosis in Oku-BL cells and derived cell clonesa
Cell line % Apoptotic cells (± SD) with: Control Staurosporine VT-1 Oku-BL 17 ± 8 63 ± 9 69 ± 2 Oku clone 1 19 ± 10 60 ± 10 72 ± 1 Oku clone 4 18 ± 9 36 ± 7 52 ± 1 Sal-BL 20 ± 2 61 ± 2 81 ± 1 ↵ a The cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells.
