Comparative analysis of p73 and p53 regulation and effector functions

Abstract

p53 is mutated in approximately 50% of human cancers, whereas mutations of the related p73 gene are rare. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. We show that p73 isoforms, p73alpha and p73beta, can each induce permanent growth arrest with markers of replicative senescence when overexpressed in a tetracycline-regulatable manner in human cancer cells lacking functional p53. Human homologue of mouse double minute 2 gene product (hMDM2), but not an NH(2)-terminal deletion mutant, coimmunoprecipitated with p73alpha or p73beta, and inhibited p73 transcriptional activity as with p53. In contrast to p53, ectopically expressed hemagglutinin (HA)-tagged p73 proteins were not stabilized by treatment with several DNA damaging agents. Furthermore, unlike normal p53, which increases in response to DNA damage due to enhanced protein stability in MCF7 cells, endogenous p73 protein levels were not increased in these cells under the same conditions. Thus, although p73 has an ability, comparable to that of p53, to suppress tumor cell growth in p53-deficient cells, p73 induction is regulated differently from p53. These findings suggest that the selective pressures for p53 rather than p73 inactivation in tumors may reflect their differential responses to stresses such as DNA damage, rather than their capacities to induce permanent growth arrest or apoptosis programs.

Figure 1

Tet regulation of p73α and p73β expression and induction of target genes in EJ-p73α (A) and EJ-p73β (B) cells. Immunoblot analysis of HA-tagged p73α and p73β as well as endogenous p21 and mdm2 expression in (+) tet (lane 1) or (−) tet for 1 d (lane 2), 2 d (lane 3), 3 d (lane 4), 4 d (lane 5), or in (−) tet for 1 d followed by the addition of tet for 1 d (lane 6).

Figure 2

Morphological changes induced by overexpression of p73α or p73β in EJ-p73α and EJ-p73β cells. EJ-p73α or EJ-p73β cells were seeded directly in (+) tet or (−) tet medium at 10⁵ cells per 100-mm plate. At day 2, 4, 6, and 8, cells were photographed using Nikon Eclipse TE200 microscope (100×).

Figure 3

Irreversible growth inhibition of EJ-p73α and EJ-p73β cells after induction of p73α or p73β. EJ-p73α or EJ-p73β cells were seeded directly in (+) tet or (−) tet at 100 cells per 60-mm plate for indicated time period. After readdition of tet for another 2 wk, colonies visualized by Giemsa staining were counted and plotted. The percentage of recoverable colonies for each condition was normalized to the number in (+) tet. All time points were performed in triplicate.

Figure 5

SA-β-gal staining after p73α or p73β induction. EJ-p73α or EJ-p73β cells were maintained in (+) tet or (−) tet medium. Cells were washed, fixed, and stained at pH 6.0 as described in Materials and Methods. Cells were photographed using a Nikon Eclipse TE200 microscope (200×). (A) EJ-p73α cells, (+) tet, 7 d. (B) EJ-p73α cells, (−) tet, 7 d. (C) EJ-p73β cells, (−) tet, 7 d.

Figure 4

Cell cycle analysis of EJ-p73α and EJ-p73β cells after induction of p73α or p73β. Cell cycle analysis was performed as described in Materials and Methods. BrdU uptake, as measured by FITC fluorescence, is depicted on the y-axis. DNA content, as measured by propidium iodide fluorescence, is depicted on the x-axis. Populations of cells in different phases of the cell cycle are gated: G0/G1 population (R1), S-phase population (R2), and G2/M population (R3). The percentage of cells in each gate is indicated for each sample in the table. (A) EJ-p73α, (+) tet. (B) EJ-p73α, (−) tet, 3 d. (C) EJ-p73β, (+) tet. (D) EJ-p73β, (−) tet, 3 d.

Figure 6

Comparison of wild-type and ΔN-hMDM2 interactions with p73α or p73β. 293T cells were transfected with HA-tagged p73α or p73β, together with pcDNA3 vector, hMDM2, or ΔN-hMDM2 as indicated at the top of each lane. Western blot analysis was performed either directly with total lysates (A) or after immunoprecipitation with antibodies as indicated (B).

Figure 7

Wild-type but not NH₂-terminal deleted hMDM2 inhibits transcriptional activities of p73α and p73β. H1299 cells in 60-mm plates were cotransfected with the plasmids indicated, as well as the p21 genomic promoter fused to a luciferase reporter and a β-galactosidase expression vector. At 48 h after transfection, luciferase activity was measured and the relative units were plotted. Results of one representative experiment of three independently performed assays are shown. Determinations were made in duplicate for each experimental point.

Figure 8

p73α and p73β proteins are not stabilized by DNA damage. (A) EJ-p53, EJ-p73α, and EJ-p73β cells were seeded in medium containing indicated tet concentrations. After 8 h, cells were treated with DNA damaging agents as indicated at the top of each lane. 24 h later, lysates were prepared and subjected to SDS-PAGE. Western blot analysis was performed using anti-p53 and HA antibodies. (B) MCF7 cells were treated with DNA damaging agents as indicated at the top of each lane. 24 h later, lysates were subjected to SDS-PAGE. Western blots were performed using anti-p53 or p73 antibodies. EJ-p73α and EJ-p73β in (−) tet were used as positive controls.