Correspondence to: Stuart A. Aaronson, Derald H. Ruttenberg Cancer Center, Box 1130, Mount Sinai School of Medicine, New York, NY 10029. Tel:(212) 659-5400 Fax:(212) 987-2240 E-mail:aaronson{at}smtplink.mssm.edu.
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Abstract |
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p53 is mutated in ~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, p73 and p73ß, 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 NH2-terminal deletion mutant, coimmunoprecipated with p73
or p73ß, 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.
Key Words: p53, p73, tumor suppression, replicative senescence, DNA damage
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Introduction |
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THE p53 gene is the most frequently inactivated tumor suppressor identified in human tumors. Approximately 50% of all human cancers lack a wild-type p53 allele, and thus fail to produce a normal version of the p53 protein (
Recently, several members of the p53 family have been identified ( and p73ß. p73ß, which is 499 amino acids (aa) in length with a unique pentamer at its extreme COOH terminus, is 137 aa shorter than p73
, which has 636 aa. In addition, p73 not only shares a high degree of similarity with p53 in primary sequence (~60% identity in the core DNA binding domain, 29% identity in NH2-terminal transactivation domain, and 42% identity in the COOH-terminal oligomerization domain), but also seems to exhibit similar functions. Like p53, both p73
and p73ß can bind to DNA and activate transcription (
in yeast two-hybrid assays (
and p73ß can block cell proliferation and induce apoptosis in cells, irrespective of their p53 status (
It is well-established that oncoproteins encoded by certain DNA tumor viruses inhibit the function of p53 (
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Materials and Methods |
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Cell Culture
The EJ human bladder carcinoma cell line, 293T human embryonic kidney cell line, and MCF7 human breast carcinoma cell line were maintained in DMEM supplemented with 10% FBS (GIBCO BRL). H1299 human lung carcinoma cells were maintained in RPMI 1640 supplemented with 10% FBS. Both EJ-p53 and EJ-p73 cells were maintained in DMEM supplemented with 10% FBS, penicillin-streptomycin (50 U/ml), hygromycin (100 µg/ml), and geneticin (750 µg/ml). To repress the expression of p73, p73ß, or p53, tet was added to the medium every 3 d to a final concentration of 1 µg/ml. To induce p73 expression, cells were washed three times with PBS and seeded directly in culture medium without tet.
Plasmid Construction and DNA Transfection
The NH2-terminal hemagglutinin (HA)-tagged coding sequence of p73 (or p73ß) (obtained from M. Kaghad, Sanofi Recherche, Innopole, France) was released with BamHI and StuI from pcDNA3-p73
(or p73ß) and then ligated with pBluescript SK digested with BamHI and EcoRV. The resulting plasmid pBluescript-p73
(or p73ß) was then digested with BamHI and SalI, and the fragment encoding p73
(or p73ß) was cloned downstream of the tet-regulated promoter into pUHD10-3 (generously provided by H. Bujard, Universitat Heidelberg, Heidelberg, Germany), resulting in plasmid pTet-p73
(or p73ß). EJ-tTA cells, generated as described previously (
(or p73ß) using the standard calcium phosphate method. Transfectants were doubly selected in the presence of hygromycin (100 µg/ml) and geneticin (750 µg/ml). Individual clones of stable transfectants, designated EJ-p73
or EJ-p73ß, were selected for further analysis.
Immunoblot Analysis
Cells cultured in the presence or absence of tet were washed twice with ice-cold PBS with 2 mM sodium vanadate and lysed in EBC lysis buffer as described previously (
Immunoprecipitation Analysis
293T cells were transiently cotransfected with p73 or p73ß and mdm2 using Fugene 6 (Boehringer Mannheim). Cell lysates were made using EBC lysis buffer and 200 µg of cellular protein was incubated with HA or MDM2 antibody at 4°C for 1 h, followed by another hour incubation with protein A beads. Immunoprecipitation complexes were washed three times with NET-N buffer (
Senescence-associated ß-Galactosidase (SA-ß-gal) Staining
Cells were cultured in the presence or absence of tet for the indicated times, washed in PBS, and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min at room temperature. The method for senescence-associated ß-galactosidase (SA-ß-gal) (pH 6.0) staining was performed as described (
Cell Cycle Analysis
Subconfluent cultures were pulse labeled for 30 min with 10 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma). Both adherent and floating cells were harvested, fixed in 70% ethanol, and then double stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody (Becton Dickinson) and 5 µg/ml propidium iodide (Sigma Chemical Co.). Cell cycle analysis was performed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson). Data were analyzed using Elite software (Becton Dickinson).
Treatment with DNA Damaging Agents
EJ-p53, EJ-p73, and EJ-p73ß cells were seeded in the presence of 2 ng/ml tet to induce submaximal levels of either p53 or p73. Cells were then treated with 2 or 5 µg/ml mitomycin C, 0.02 or 0.1 µg/ml doxorubicin, or 5 or 10 ng/ml actinomycin D for 24 h. MCF7 cells were treated under the same condition. Cell lysates were prepared and aliquots containing 40 µg of cell protein were subjected to SDS-PAGE followed by immunoblot analysis with 1801 mAb for p53, HA polyclonal antibody, or ER-15 mAb for p73.
Luciferase Assays
Plasmid DNA was transiently transfected into H1299 cells using Fugene 6 (Boehringer Mannheim). Approximately 2 x 106 cells were cotransfected with plasmids as indicated. Cells were harvested 48 h after transfection, and luciferase activity was measured using a luciferase assay kit (Promega). The assay was normalized by cotransfection of a pCMVß-gal plasmid and measurement of ß-galactosidase activities.
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Results |
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Inducible Expression of p73 or p73ß in EJ Cells
It has been shown that p53 can induce growth arrest, apoptosis, or senescence depending on the cell context ( or p73ß expression, the tet-regulatable expression system (
or pTet-p73ß containing HA-tag and a neomycin-resistant marker. Stable clones were isolated by double selection. More than 10 tet-regulatable clones for each gene were selected and two independent clones were analyzed in detail. The phenotypes in each case (designated EJ-p73
or EJ-p73ß) were similar. As shown in Figure 1 A, there was no detectable amount of HA-p73
in the presence of 1 µg/ml tet as determined by immunoblot analysis (Figure 1 A, lane 1). Within 24 h of tet removal, p73
was induced and became readily detectable (Figure 1 A, lane 2), with p73
levels further increasing to a steady-state level by 48 h (Figure 1 A, lane 3). To test whether p73
induction was reversible, tet was added back to the medium after induction for 24 h, and p73
levels examined 24 h later. It was apparent that p73
returned to an undetectable level (compare lanes 6 and 2 in Figure 1 A) under these conditions, indicating that p73
expression was fully reversible. Similar results were obtained with EJ-p73ß as shown in Figure 1 B.
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It can also been observed that two p53 transcriptional target genes, p21 and mdm2, were induced by both p73 and p73ß (Figure 1). The kinetics of the induction paralleled that of p73, and was also reversible following readdition of tet (Figure 1A and Figure B, lanes 6).
p73 Induces Irreversible Growth Arrest Associated with Senescence-like Morphology
In response to p73 induction in EJ-p73 or EJ-p73ß cells, we observed profound alterations in both cell proliferative capacity and morphology. Whereas EJ-p73 cells grew as small, rounded, refractile cells in the presence of tet and reached confluence, similar to parental EJ cells, the induction of p73 expression caused cells to stop growth and exhibit increased size and flattened morphology as well as enlarged nuclei (Figure 2). Of note, there were no characteristics of apoptosis detected in these cells as determined by 4',6-diamidino-2'-phenylindole dihydrochloride nuclear staining (data not shown). To examine the reversibility of p73-induced growth arrest in EJ cells, we performed a colony formation assay. EJ-p73
or EJ-p73ß cells were seeded at about 100 cells per 60-mm plate and maintained in the absence of tet for varying time periods followed by tet readdition. Cultures were subsequently maintained in the presence of tet for another 2 wk, followed by fixation and Giemsa staining. The number of colonies were counted and plotted as shown in Figure 3. Maintenance of the cells in the absence of tet for three or more days resulted in a marked reduction of the ability to form colonies. Indeed, the kinetics of permanent inhibition of colony formation by p73
or p73ß was comparable to that observed with p53 (
or p73ß in EJ cells causes irreversible growth arrest.
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Expression of p73 Induces G1 and G2 Cell Cycle Arrest
To investigate in which specific cell cycle stage(s) p73 arrested EJ cells, we performed fluorescence-activated cell sorting analysis using EJ-p73 or EJ-p73ß cell. EJ-p73 cells were maintained in the presence or absence of tet for varying time periods, followed by analysis using simultaneous flow cytometry for both DNA content and DNA synthesis, with propidium iodide staining and BrdU labeling, respectively. After tet removal, EJ-p73 cells exhibited a dramatic reduction in BrdU incorporation within 3 d, with the population of S phase cells declining from 45.2 and 51.7% in (+) tet to 5.9 and 12.4% in (-) tet for EJ-p73
or EJ-p73ß, respectively (Figure 4). Conversely, the percentage of cells in both G1 and G2/M phases increased from 34.1 and 20.2% in (+) tet to 60.5 and 33.6% in (-) tet for EJ-p73
, and from 31.5 and 16.0% in (+) tet to 56.3 and 26.5% in (-) tet for EJ-p73ß by 3 d, respectively. Thus, induced expression of p73
or p73ß arrested EJ cells in both G1 and G2/M phases. Induction of both G1 and G2/M arrest has also been observed with p53 overexpression in EJ-p53 cells (
or ß cells.
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Expression of a Senescence-specific Marker after p73 Induction
It has been shown that senescent but not presenescent, quiescent, or terminally differentiated cells express a SA-ß-gal, which can be detected by incubating cells at pH 6.0 with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) ( or p73ß, whereas EJ-p73 cells grown in the presence of tet over the entire time course of the experiment showed no staining (only [+] tet 7 d of EJ-p73
is shown). These results indicated that expression of p73 can promote a senescence-like program in EJ cells.
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Wild-Type but Not NH2-Terminal Deleted hMDM2 Interacts with p73 or p73ß and Inhibits Their Transcriptional Activity
It has been reported that the product of mdm2, a p53 transcriptional response gene, can interact with p53 and target it for degradation ( or p73ß, together with wild-type or a mutant human MDM2 with the first 58 aa deleted (
N-hMDM2). This deletion is known to abolish MDM2's ability to interact with p53 (
or p73ß was detected in the immunocomplexes precipitated by the anti-mdm2 antibody; similarly, hMDM2 was also detected in the immunocomplexes precipitated by the anti-HA antibody. However, there was no detectable p73
or p73ß associated with the mutant hMDM2; similarly, mutant hMDM2 was not detected in the immunocomplexes associated with p73
or p73ß. These experiments demonstrated that p73
and p73ß interact with wild-type but not NH2-terminal deleted hMDM2, despite the comparable expression level of these proteins (Figure 6 A).
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Next, we attempted to investigate whether the interaction between hMDM2 and p73 had any effects on p73's transcriptional activity. To do so, p73 or vector was cotransfected into H1299 cells along with a luciferase reporter plasmid that contains the genomic sequence from the p21 promoter. As shown in Figure 7, p53, p73, and p73ß increased the luciferase activity by 15-, 18-, and 38-fold compared with vector, respectively. Neither hMDM2 nor
N-hMDM2 alone had any effect on the luciferase activity. When wild-type mdm2 was cotransfected with p53, p73
, or p73ß, the luciferase activity decreased three-, four-, and sixfold, respectively. However, when
N-hMDM2 was cotransfected, there was no significant change in the p21 promoter response. These experiments demonstrated that wild-type hMDM2 interacts with p73 and specifically inhibits its transcriptional activation of the p21 promoter, consistent with recent reports (
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p73 Is Not Induced at a Posttranslational Level by DNA Damaging Agents
It has been shown that p53 is induced by various stresses such as DNA damage, hypoxia, or nucleotide pool depletion ( and p73ß did not increase after exposure to any of these DNA damaging agents. Since the HA-tagged p73 was transcriptionally active, it is unlikely that it would respond differently from endogenous p73 to DNA damage, although we cannot exclude this possibility. Thus, we next tested whether endogenous p73 behaved similarly.
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MCF7 cells were treated with different concentrations of DNA damaging agents, followed by immunoblot analysis with p53 or p73 antibodies. As shown in Figure 8 B, p53 levels increased after each treatment. However, the levels of both p73 and p73ß did not increase in response to any of the DNA damaging agents tested. These results suggested that unlike p53, p73 protein stability was not increased in response to several different genotoxic agents.
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Discussion |
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These studies demonstrate that in tumor cells lacking functional p53, the induced overexpression of either p73 or p73ß, an alternative product of the p73 gene, promoted a cellular response leading to irreversible growth arrest with markers of replicative senescence. This conclusion is supported by the following observations: induction of a flattened, enlarged cell morphology, commonly observed with senescent fibroblasts; and SA-ß-gal staining (pH 6.0), a specific biochemical marker of senescent cells (
We also found that p73 can induce mdm2 and p21, two known transcriptional targets of p53, consistent with previous studies ( and p73ß, and this interaction was also disrupted by deletion of the NH2-terminal 58 aa residues of hMDM2, indicating that p73 interacts through the same NH2-terminal 58 residues. We further observed that hMDM2 inhibited the transcriptional response from the p21 promoter in response to p73, as has been reported for p53 (
Unlike the case with p53, hMDM2 interaction did not target p73 for degradation, since p73 protein levels did not decrease (Figure 6 A). These results indicate that although hMDM2 can interact with both p53 and p73, its inhibition of p73 transcriptional activity is not mediated by a mechanism involving p73 protein degradation. Similar findings have been reported recently by
We observed another major difference in p53 and p73 biology. In EJ tumor cells in which p53 function had been inactivated, exogenously expressed p53 but not p73 showed increased protein level in response to several different DNA damaging agents. Since transcription of each gene was under the control of the same tet-regulatable promoter, these findings likely reflect p53 protein stabilization in response to genotoxic stress by mechanisms that remained intact in these tumor cells. The lack of response of p73 to the same agents further implies differential regulation of these genes at the level of protein stabilization in these tumor cells. These findings could help to explain a selective pressure for inactivation of p53 but not p73 function in the evolution of this tumor despite their comparable ability of inducing permanent growth arrest in these cells.
We also observed that in MCF7 breast cancer cells with intact p53, neither endogenous p73 nor p73ß was induced by DNA damaging agents under conditions in which p53 overexpression was readily observed, consistent with a previous report (
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Footnotes |
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1 Abbreviations used in this paper: aa, amino acid(s); BrdU, 5-bromo-2'-deoxyuridine; HA, hemagglutinin; hMDM2, human homolgue of MDM2; MDM2, mouse double minute 2 gene product; SA-ß-gal, senescence-associated ß-galactosidase; tet, tetracycline.
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Acknowledgements |
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We thank Dr. Mourad Kaghad for kindly providing p73 plasmids and Dr. Zhenqiang Pan for critical reading of the manuscript.
This work was supported in part by National Institutes of Health grant CA66654 and the T.J. Martell Foundation for Leukemia Cancer and AIDS Research (to S.A. Aaronson), and National Institutes of Health grants CA78356 and CA82211 (to S.W. Lee). L. Fang is a recipient of Forchheimer Foundation Fellowship.
Submitted: 21 July 1999
Revised: 30 September 1999
Accepted: 4 October 1999
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References |
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