Figure 1.
Expression of TSC-22 was decreased in human cancer tissue.
(A) Total RNA was prepared from patients’ cancer specimens and the TSC-22 mRNA level was then evaluated with real-time RT-PCR. Cx; serial number of patient tissue samples. (B) HeLa and Caski cells were plated on 6-well plates. After 24 h, the cells were infected with Ad-TSC-22 or Ad-LacZ. At the indicated time points, cell numbers were determined by MTT assay to analyze cell proliferation rates. (C) HeLa cells infected with Ad-TSC22 or Ad-LacZ were cultured for the indicated times, and the cells were then stained with propidium iodide (PI). The sub-G1 cell population (dead cell) and cell cycle profile of the PI-stained cells were analyzed by flow cytometry after PI-staining. (D) DNA fragmentation assay was performed by isolating chromosomal DNA from 1×106 number of HeLa and Caski cells infected with Ad-TSC22 or Ad-lacZ for 72 hrs (left). After 3 days infection of Ad-TSC-22, p53, Puma, p21, E6 and TSC22 expression were analyzed by Western hybridization (right).
Figure 2.
TSC-22 induces p53 expression.
(A) TSC-22 and p53 cDNA constructs were cotransformed into EGY48 yeast cells to test for protein–protein interaction within the yeast two-hybrid system. Transformants were assayed for their ability to grow on medium lacking leucine (left) and for β-galactoside expression (right). (B) HeLa cells and Caski cells were infected with Ad-TSC-22 for the indicated times. Protein levels were analyzed by Western blot with the DO-1 antibody against p53, the anti-TSC-22 antibody, and the anti-β-actin antibody as a loading control. (C) HeLa cells were transfected with 1 µg of Flag-TSC-22 expression vector. 24 h post-transfection, p53, Puma, p21, and TSC22 expression were analyzed by Western blotting and semi-quantitative RT-PCR with protein and total RNA obtained from each cell line. (D) HeLa cells stably expressing shRNA specific for TSC-22 and non-targeting control shRNA were analyzed to determine the protein and mRNA expression levels of p53 and Puma. (E) Luciferase activity of the p53RE (responsible element)-driven promoter were assessed with transfection of the indicated amount of Flag-TSC-22 plasmid in HeLa cells (upper panel). Activity of the p53RE-promoter was assessed in sh-con and sh-TSC-22 expressing HeLa cells (lower panel) by luciferase assays. (F) One µg of Flag-TSC-22 or Flag-mock vector was transfected into p53+/+ or p53−/− HCT116 cells. At 48 h post-transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. (G) Stability of p53 protein was assessed in HeLa cells infected with Ad-TSC-22 or Ad-LacZ. 24 h after infection, cells were treated with cycloheximide (50 µg/mL) for the indicated periods of time. Cell lysates were analyzed by Western blotting with anti-p53 antibody (DO-1) with β-actin as a loading control.
Figure 3.
TSC22 interacts with p53 in vivo. (A–B) TSC22 interacts with ectopic p53.
Ectopically expressed TSC-22 interacts with ectopically expressed p53 in H1299 cells. Two µg of Flag-TSC-22 expression vector was cotransfected with Flag-p53 expression vector (A) or a non-tagged p53 expression vector (B) into H1299 cells cultured in 100 mm plates. 48 h after transfection, cell lysates were immunoprecipitated with an anti-p53 (DO-1) (A) or anti-Flag (B) monoclonal antibody. Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-Flag (A) or anti-p53 (FL393) (B) monoclonal antibody. (C–D) TSC-22 interacts with endogenous p53. Ectopically expressed TSC-22 interacts with endogenous p53 in cells. HEK293 cells were transfected with 2 µg of Flag-TSC-22 plasmid for 48 h. Cell lysates were immunoprecipitated with anti-p53 (DO-1) monoclonal antibody, mouse immunoglobulin G (IgG) (C), or an anti-Flag (D) monoclonal antibody. Immunoprecipitates were analyzed with Western blotting using an HRP-conjugated anti-Flag (C) or anti-p53 (FL393) antibody.
Figure 4.
Mapping of the binding regions in TSC-22 and p53.
(A) Schematic diagram shows the cDNA constructs for the Flag-tagged p53 deletion mutants and full-length p53, and indicates the TSC22 binding domain. (B) H1299 cells were transfected with the indicated plasmids encoding Flag-tagged p53 deletion mutants along with the Flag-TSC-22 plasmid. Cell lysates were immunoprecipitated with the anti-p53 polyclonal antibody FL393 (left) or DO-1 monoclonal antibody specific for the N-terminal region (right), followed by Western blotting using the indicated antibodies. Lysates (5%) were also loaded onto an SDS gel for Western blotting using an anti-Flag antibody (bottom of each panel). (C) Schematic diagram showing the cDNA constructs of the Flag-tagged TSC-22 deletion mutants (left panel). H1299 cells were transfected with the indicated plasmids encoding the Flag-tagged TSC22 deletion mutants together with Flag-p53 plasmid. Cell lysates were immunoprecipitated with an anti-p53 antibody (FL393); co-immunoprecipitated TSC22 was detected by Western blotting using the HRP-conjugated anti-Flag antibody (right, middle panel). Lysate (5%) was analyzed by Western blotting using an anti-Flag antibody (right, bottom panel).
Figure 5.
TSC22 inhibits HDM2- and E6-mediated p53 ubiquitination.
(A) TSC22 inhibits HDM2-mediated p53 ubiquitination. H1299 cells were transfected with the indicated plasmids. The transfected cells were treated with MG132 (20 µM) for 5 h before harvest. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, HDM2, Flag-TSC-22 and HA-Ub proteins are shown in the lower panels. (B) TSC-22 does not interrupt interaction between p53 and HDM2. H1299 cells were transfected with Flag-p53 along with Flag-TSC-22 or a Flag-mock vector in the presence of an HDM2 expression vector. Cell lysates were immunoprecipitated with the anti-p53 (DO-1) antibody followed by Western blotting using the indicated antibodies. Lysate (5%) was analyzed by Western blotting using indicated antibodies (lower panel). (C) TSC-22 inhibits E6-mediated p53 ubiquitination. H1299 cells were transfected with the indicated plasmids in presence of an HA-Ub expression vector. 48 h after transfection, the transfected cells were treated with MG132 (20 µM) for 5 h before they were harvested. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with the anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, Myc-E6, Flag-TSC-22, and HA-Ub proteins are shown in the lower panels. (D) TSC-22 disrupts ubiquitination of p53 in HeLa cells. HeLa cells were cotransfected with Flag-TSC-22 or a Flag-mock vector and HA-Ub expression vector. 48 h after transfection, cells were treated with 20 µM MG132 for 5 h prior to harvesting. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). (E) Stable TSC-22 knock-down or control HeLa cells were treated with 20 µM MG132 for 5 h prior to harvesting. Ubiquitination of p53 was analyzed as described above.
Figure 6.
TSC-22 inhibits tumor growth in nude mice.
(A) HDM2 and Myc-E6 were transfected with indicated plasmids into H1299 cells. The transfected cells were treated with MG132 (20 µM) for 5 h before harvest. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, HDM2, Flag-TSC-22 and HA-Ub proteins are shown in the lower panels. (B) Flag-tagged wild type (WT) or mutant TSC221–110 was co-transfected with the indicated plasmids into H1299 cells. The transfected cells were treated with MG132 (20 µM) for 5 h before harvest. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, HDM2, Flag-TSC-22 and HA-Ub proteins are shown in the lower panels. (C) Nude mice were inoculated with 1×106 HeLa cells by subcutaneous injection. Subcutaneous tumors derived from the HeLa cells were treated with adenovirus vectors as indicated. Tumor volumes are shown as the mean from at least five mice per group (n = 10 to 14 per group). Bars = SD. (D) Effect of TSC-22 treatment on the expression level of p53 in HeLa cell-derived tumors excised at the 27th day post-treatment as determined by immunoblot analysis. Representative immunoblot of the three samples from each group.