Recombinant replication-deficient adenovirus delivery of ZBP-89. AGS cells were mock infected or infected with the control Ad5-vector (Ad-vector) or Ad5-ZBP-89 (Ad-ZBP-89) at MOIs of 2 and 10. Forty-eight hours later, cells were collected, lysed, and separated by SDS-PAGE. The monoclonal Flag M2 and polyclonal ZBP-89 antibodies were used to detect Flag-tagged and endogenous ZBP-89 proteins. A monoclonal actin antibody was used to control for the amount of protein loaded.

ZBP-89 induces wild-type p53 protein accumulation. (A) AGS cells were mock infected or infected with the indicated recombinant adenoviruses. The protein expression levels of Flag-tagged ZBP-89, wild-type p53, procaspase-3, and p21^waf1 were measured by immunoblotting. Lane 1, mock infection; lane 2, Ad5-vector; lane 3, Ad5-ZBP-89. (B) ZBP-89 and induced p53 expression colocalizes to the nucleus. The AGS cells were infected with control Ad5-β-gal or Ad5-ZBP-89 at an MOI of 10. Forty-eight hours after infection, the cells were immunostained with mouse anti-p53 (green) and rabbit anti-ZBP-89 (red) antibodies. Left, Ad5-β-gal-infected cells; right, Ad5-ZBP-89-infected cells. DAPI was used to stain the nuclei (blue). Yellow indicates colocalization of ZBP-89 and p53, which were photographed with a dual filter. (C) Flow cytometry analysis of HCT 116 wild-type and p53 null cells. Mock-infected, Ad5-vector, or Ad5-ZBP-89 recombinant adenoviruses were used infect HCT 116 wild-type and p53 null cells at an MOI of 40 as described above prior to flow analysis. (D) The sub-G₁ population was determined by flow cytometry as described above. Shown are the means ± standard errors of the means for three experiments.

ZBP-89 stabilizes the p53 protein. (A) AGS and HCT 116 cells were infected with control and ZBP-89-expressing adenovirus vectors at an MOI of 10 for 2 days. Total RNA was prepared for Northern blot analysis. The blot was first probed with a p53 riboprobe (top) and then stripped and reprobed for GAPDH (bottom). Lanes 1 and 4, mock infection; lanes 2 and 5, Ad5-β-gal; lanes 3 and 6, Ad5-ZBP-89. (B) AGS cells were infected with the control Ad5-β-gal or Ad5-ZBP-89 for 16 h. The half-life of p53 was then determined by [³⁵S]methionine pulse-chase analysis. The p53 protein was immunoprecipitated with mouse anti-p53 antibody (DO-1) and then resolved on a NOVEX 4 to 12% gradient gel. (C) The p53 band intensity was quantified by PhosphorImager analysis. Band intensities were expressed as percentages of the control signal (pulse only, no chase). Results shown are representative of three experiments.

ZBP-89 enhances p53 transcriptional activation. (A) HCT 116 p53^−/− cells were cotransfected with 200 ng of PG13 or MG15, 5 ng of pCMV-p53, and/or 100 ng of pcDNA3-Flag-ZBP-89. Relative luciferase activities were obtained by normalizing the luciferase activity with β-galactosidase activity. Values are the means ± standard errors of the means (SEM) for three independent experiments performed in triplicate. (B) HCT 116 p53^−/− cells were cotransfected with 200 ng of p21^waf1-Luc, 5 ng of pCMV-p53, and/or 100 ng of pcDNA3-Flag-ZBP-89. Relative luciferase activities were obtained by normalizing the luciferase activity with β-galactosidase activity. Shown are the means ± SEM for three experiments performed in triplicate. (C) Immunoblot analysis of transiently transfected HCT 116 p53^−/− cells. Eighty micrograms of whole-cell extracts was resolved by SDS-PAGE. The anti-β-galactosidase, anti-p53, and anti-Flag M2 monoclonal antibodies were used. Results shown are representative of three independent experiments.

ZBP-89 stabilizes p53 without interrupting the p53-MDM2 interaction. (A) AGS cells were infected, and an immunoblot was performed, as described in the legend for Fig. 1. The polyclonal p53, ZBP-89, actin, and monoclonal Flag M2 and MDM2 antibodies were used. Lane 1, mock infection; lane 2, Ad5-β-gal; lane 3, Ad5-ZBP-89. (B) AGS whole-cell extracts were immunoprecipitated with the mouse p53 antibody (DO-1). The pellets were resolved on a NOVEX 4 to 12% gel followed by immunoblotting as described in the legend for Fig. 7. Lane 1, mock infection; lane 2, Ad5-β-gal; lane 3, Ad5-ZBP-89.

ZBP-89 induces growth arrest and apoptosis in AGS cells. (A) AGS cells were mock infected or infected with the indicated recombinant adenovirus (vector alone [Ad-vector] or ZBP-89 [Ad-ZBP-89]) at an MOI of 10. Two days later, cells were collected, fixed, and stained with propidium iodide. The DNA content was quantified using a FACSCaliber. Shown are the means of three experiments performed in triplicate ± standard errors of the means (SEM). (B) AGS cells were transiently transfected with the pcDNA3 vector or pcDNA3-Flag-ZBP-89 and then labeled with BrdU as described in Materials and Methods. DAPI was used to stain the nuclei (blue). Rabbit anti-ZBP-89 (followed by Texas red-conjugated anti-rabbit IgG) and FITC-conjugated anti-BrdU antibodies were used to stain ZBP-89 (red) and BrdU (green), respectively. The results shown are representative of three different experiments. (C) The presence of apoptotic cells in AGS cells was determined by the TUNEL assay. DAPI was used to stain the nuclei (blue). The immunolocalization of ZBP-89 was as described for panel B. FITC-conjugated dUTP labeled apoptotic cells green (TUNEL). Yellow indicates the colocalization of the ZBP-89 protein with apoptotic events in the same cell photographed with a dual filter on an Olympus BX60. Results shown are representative of three experiments. (D) The populations of sub-G₁ cells from mock infection and each recombinant adenovirus-infected cell population were determined by flow cytometry. Shown are the means of three experiments ± SEM.

Serum starvation induces ZBP-89 and p53 expression. (A) AGS cells were cultured in serum-free F-12 medium for the indicated times, and immunoblots were used to detect the expression profiles of ZBP-89, p53, and p14ARF. (B) AGS cells were grown in serum-free medium for 24 h, and 200 μg of nuclear extracts was used for immunoprecipitation (IP). Lanes 1 and 2, immunoprecipitation with mouse anti-p53 antibody (DO-1) at 0 and 24 h after serum starvation; lane 3, immunoprecipitation with control mouse IgG at 24 h. Blotting was with rabbit anti-ZBP-89 or anti-p53 antibodies.

ZBP-89 interacts with p53 in vivo and in vitro. (A) Coimmunoprecipitation of p53 and ZBP-89 from mock-infected AGS cells (lane 1) or from cells infected with Ad5-β-gal (lane 2) or Ad5-ZBP-89 (lane 3). After cell lysis, immunoprecipitation (IP) was performed with the polyclonal p53 antibody followed by immunoblotting with p53 and Flag M2 antibodies. (B) Immunoprecipitation of ZBP-89 and p53 was performed with in vitro-transcribed and -translated ZBP-89 and p53 proteins. Lane 1, input of [³⁵S]methionine-labeled ZBP-89; lane 2, [³⁵S]methionine-labeled p53; lane 3, unlabeled ZBP-89 incubated with radiolabeled p53 and immunoprecipitated with control rabbit serum; lane 4, unlabeled ZBP-89 incubated with radiolabeled p53 and immunoprecipitated with rabbit ZBP-89 antibody; lane 5, unlabeled p53 incubated with radiolabeled ZBP-89 and immunoprecipitated with control rabbit serum; lane 6, unlabeled p53 incubated with radiolabeled ZBP-89 and immunoprecipitated with rabbit anti-p53 antibody. PI, preimmune serum. (C) Mapping of the p53-interacting domain of ZBP-89. The various ZBP-89 constructs used in GST pull-down assays are shown. The ZBP-89 GST fusion proteins were incubated with [³⁵S]methionine-labeled, in vitro-translated p53, pelleted, and then analyzed by SDS-PAGE. Lane 1, input of p53; lane 2, GST alone. (D) Mapping of the ZBP-89-interacting domain of p53. The various p53 constructs used in GST pull-down assays are shown. These proteins were incubated with [³⁵S]methionine-labeled, in vitro-translated ZBP-89 and analyzed as described above for p53. Lane 1, input of labeled ZBP-89; lane 2, GST alone. (E) GST pull-down was performed in the presence of 10 μg of ethidium bromide/ml, and blotting was performed with the M2 anti-Flag antibody. (F) Schematic representation of the ZBP-89 and p53 interaction domains. ZBP-89 interacts with amino acids 160 to 393 of p53. p53 contains an activation domain (AD), DNA binding domain (DNA BD), tetramerization domain (TD), and regulatory domain (RD). p53 binds to the zinc finger DNA binding domain of ZBP-89 (amino acids 154 to 300). ZBP-89 contains an acidic domain (AD), zinc finger DNA binding domain, basic domain (BD), and C-terminal domain (C-TER).

The p53R273H mutation prevents ZBP-89-mediated stabilization. (A) HCT 116 (wild-type p53 and null) and HT-29 colon cell lines were treated as described in the legend for Fig. 1. The expression of ZBP-89 and p53 was determined by immunoblot analysis. Lanes 1, 4, and 7, mock infection; lanes 2, 5, and 8, Ad5-vector; lanes 3, 6, and 9, Ad5-ZBP-89. (B) Two hundred micrograms of HT-29 whole-cell extracts was incubated with 20 μl of GST or GST–ZBP-89 beads at 4°C for 1 h. The pellets were washed; proteins were eluted from beads and resolved by SDS-PAGE for immunoblot analysis. Twenty micrograms of whole-cell extracts was loaded as the input. (C) HCT 116 p53^−/− cells were cotransfected with 200 ng of PG13 or p21^waf1-Luc, 5 ng of pCMV-p53R273H, and/or 100 ng of pcDNA3-Flag-ZBP-89. Relative luciferase activities were obtained by normalizing the luciferase activity with β-galactosidase activity. Values are means ± standard errors of the means from three independent experiments performed in triplicate. (D) Western blot analysis of transiently transfected HCT 116 p53^−/− cells. Eighty micrograms of whole-cell extracts was separated by SDS-PAGE for immunoblot analysis.

ZBP-89 prevents the nuclear export of p53. AGS cells were transfected with the pcDNA3 empty vector or the pcDNA3-Flag-ZBP-89 expression vector. Forty hours after transfection, the heterokaryon assay was performed as described in Materials and Methods. Phase-contrast optics were used to confirm the fusion of AGS (arrows) with MEF p53 null cells. The nucleus was stained with DAPI and visualized using a UV filter (blue). p53 was stained with a FITC-labeled anti-mouse IgG (green), and ZBP-89 was stained with a Texas red-conjugated anti-rabbit IgG (red).