PTTG/securin activates expression of p53 and modulates its function
© Hamid and Kakar; licensee BioMed Central Ltd. 2004
Received: 26 May 2004
Accepted: 08 July 2004
Published: 08 July 2004
Pituitary tumor transforming gene (PTTG) is a novel oncogene that is expressed abundantly in most tumors. Overexpression of PTTG induces cellular transformation and promotes tumor formation in nude mice. PTTG has been implicated in various cellular processes including sister chromatid separation during cell division as well as induction of apoptosis through p53-dependent and p53-independent mechanisms. The relationship between PTTG and p53 remains unclear, however.
Here we report the effects of overexpression of PTTG on the expression and function of p53. Our results indicate that overexpression of PTTG regulates the expression of the p53 gene at both the transcriptional and translational levels and that this ability of PTTG to activate the expression of p53 gene is dependent upon the p53 status of the cell. Deletion analysis of the p53 gene promoter revealed that only a small region of the p53 gene promoter is required for its activation by PTTG and further indicated that the activation of p53 gene by PTTG is an indirect effect that is mediated through the regulation of the expression of c-myc, which then interacts with the p53 gene promoter. Our results also indicate that overexpression of PTTG stimulates expression of the Bax gene, one of the known downstream targets of p53, and induces apoptosis in a human embryonic kidney cell line (HEK293). This stimulation of bax expression by PTTG is indirect and is mediated through modulation of p53 gene expression.
Overexpression of PTTG activates the expression of p53 and modulates its function, with this action of PTTG being mediated through the regulation of c-myc expression. PTTG also up-regulates the activity of the bax promoter and increases the expression of bax through modulation of p53 expression.
The pituitary tumor transforming gene (PTTG), a securin, was cloned initially from a rat pituitary tumor . Subsequently, we and others cloned the human PTTG gene [2, 3] and characterized its function. PTTG protein is expressed at higher than normal levels in several tumors, including those of the pituitary , thyroid , colon , ovary , testis  and breast , as well as in hematopoietic neoplasms . In some tumors, including those of the thyroid , pituitary , esophagus  and colorectum , high levels of expression of PTTG correlate with tumor invasiveness and, more recently, PTTG has been identified as a key "signature gene", with high expression predicting metastasis in multiple tumor types . Overexpression of PTTG enhances cell proliferation, induces cellular transformation, and promotes tumor formation in nude mice [2, 3]. The involvement of PTTG in several cellular functions, such as mitosis [14, 15], cell cycle progression , DNA repair  and secretion and expression of basic fibroblast growth factor (bFGF)  and vascular endothelial growth factor (VEGF)  has been reported.
Structural homology suggested that PTTG may be a mammalian securin and this has been confirmed by its involvement in regulating sister chromatid separation during mitosis . The involvement of PTTG in cell signaling via the MAP kinase cascade  and its interaction with the c-myc promoter suggest its involvement in cellular transformation .
There is now considerable evidence supporting the concept that PTTG can regulate apoptosis in both a p53-dependent and a p53-independent manner . In their studies, Yu et al.  have shown that overexpression of PTTG results in the induction of apoptosis of cells that are p53 deficient (osteosarcoma MG-63 cells), as well as cells that express functional p53 (breast tumor MCF-7 cells), with overexpression of both PTTG and p53 resulting in an increase in apoptosis of MCF-7 cells but not MG-63 cells. However, overexpression of E6, which targets p53 for degradation, eliminated p53 in MCF-7 cells but did not inhibit apoptosis on overexpression of PTTG. Analysis of the molecular basis for the interaction between PTTG and p53 by these investigators  showed that overexpression of PTTG in MCF-7 cells resulted in an increase in translocation of p53 protein to the nucleus. Subsequently, Bernal et al.  using phage-display screening identified an interaction of the p53 and PTTG proteins in vitro and in vivo. These investigators found that this interaction of p53 with PTTG inhibited the transcriptional activity of p53 and its ability to induce cell death in MCF-7 cells. These investigators did not find activation of p53 expression on overexpression of PTTG in the lung tumor cell line, H1299. In these cells, overexpression of both PTTG and p53 resulted in down regulation of p53-induced apoptosis together with down regulation of the p53-induced expression of downstream signaling genes, including Bax, SFN, and CDKN1A suggesting that, in these cells, PTTG inhibits the function of p53 rather than its expression.
Our previous analysis of the PTTG promoter revealed tumor-specific activation of the promoter in various cell lines and indicated that Sp1 and NF-Y binding sites within the PTTG promoter are involved in its activation . Zhou et al.  recently confirmed that Sp1 and NF-Y are involved in the expression of PTTG and that these binding sites play an important role in the regulation of the expression of PTTG by p53. We therefore undertook an analysis of the mechanisms by which PTTG regulates p53 expression and function. Here, we report that PTTG regulates p53 expression and function by modulating the activity of the p53 promoter indirectly through regulation of the expression of c-myc, which binds directly to the p53 promoter sequence.
PTTG upregulates expression of p53 in HEK293 and MCF-7 cells
Up-regulation of p53 promoter activity by PTTG is mediated by regulation of expression of the c-myc gene
PTTG activates bax gene expression by inducing p53 expression
Overexpression of PTTG induces apoptosis
The present study was focused on determining the mechanism(s) by which PTTG regulates the expression of p53. For this purpose, we used three different cell lines: HEK293 and MCF7, which express wild-type p53; and PC-3, which expresses a mutant form of p53 that is degraded rapidly. Overexpression of PTTG led to activation of p53 expression in the HEK293 and MCF-7 cell lines, but not the control PC-3 cells (Fig. 1A). Immunohistochemical analysis of the HEK293 cells that had been transfected with pcDNA3.1-PTTG confirmed that overexpression of PTTG protein resulted in high levels of expression of the p53 protein (Fig. 1B). The activation of the expression of the p53 protein by PTTG in MCF-7 cells is consistent with the findings of Yu et al.  who reported an increase in expression of p53 and its translocation into nucleus on overexpression of PTTG in these cells. In their studies, Yu et al.  did not find simultaneous accumulation of p53 and mdm2 in the nucleus, indicating that PTTG may not use the ARF mechanism to induce p53 expression. It is possible that PTTG may induce p53 nuclear accumulation by inhibiting mdm2 expression through other mechanisms . It also is possible that enhancement of the expression of p53 may lead to the activation of other pathways, which could alter the expression of other genes or the interaction of p53 with other proteins . For example, enhanced p53 may activate bax, an antagonist of Bcl-2 , or enhance the synthesis of insulin-like growth factor-I (IGF-I) receptor and one of its binding proteins, IGF-BP3 .
The mechanisms by which PTTG up-regulates the expression of p53 may include enhancement of the stability of mRNA, an improvement in translation efficiency, or enhanced transcription of the gene. Our studies suggest that the enhanced expression of the p53 gene is due, at least in part, to an increase in p53 promoter activity. It has been shown that the PTTG possesses transactivating  and DNA binding properties . We therefore expected that PTTG might regulate p53 promoter activity by virtue of its direct binding to the p53 gene promoter sequence; however, our DNA foot printing and gel shift assays showed no direct binding of PTTG protein to the p53 promoter sequence, suggesting that PTTG modulates p53 promoter activity through an indirect mechanism. Subsequent 5' and 3' deletion analysis of the p53 gene promoter identified the sequence between nucleotides -172 to -89, which contains a c-myc/max binding sequence, as being responsive to PTTG activation. In previous studies, Pei  demonstrated binding of PTTG protein to the c-myc gene promoter sequence near its transcription start site and its activation by PTTG. Therefore, it is possible that the enhancement of expression of the p53 gene by PTTG is a result of enhancement of the expression of c-myc. The resulting c-myc would then interact with its partner, max, to form a heterodimer that binds to the p53 promoter and regulates its transcription. Binding of the c-myc protein to this sequence was confirmed by gel mobility shift and super shift assays, which revealed specific binding of the c-myc protein to this sequence. Site-directed mutagenesis of the c-myc/max sequence resulted in a complete loss of binding of the c-myc protein and p53 promoter activation by PTTG. The importance of c-myc in regulating expression of the p53 gene by PTTG is further supported by the results generated using a c-myc dominant-negative construct that abolished the induction of p53 gene expression by PTTG. These results demonstrate clearly that PTTG up-regulates the expression and transcription of the p53 gene by modulating the expression of the c-myc gene and its binding to the c-myc/max sequence on the p53 promoter.
PTTG is an oncogene and has been shown to induce cellular transformation in vitro and promote tumor formation in nude mice . In an attempt to define the mechanism by which PTTG contributes to tumorigenesis, Bernal et al.  used phage display screening and determined that the p53 protein interacts with the PTTG protein both in vitro and in vivo leading to inhibition of the transcriptional activity of p53 and its ability to induce cell death. In contrast to our results, these investigators did not find that PTTG altered the expression of p53. In their studies, these investigators showed a very low or marginal increase in the levels of PTTG protein on transfection with PTTG cDNA. Therefore, the discrepancy between Bernal's findings and ours may be attributable to the requirement for a threshold amount of PTTG protein for induction of p53 promoter activity. Zhou et al.  showed suppression of p53 expression in cells treated with the DNA-damaging drugs doxorubicin and bleomycin. This drug-induced suppression of PTTG was shown to be dependent on the presence of functional p53. In their studies, these investigators  showed direct suppression of PTTG expression by p53 through its interaction with the NF-Y transcription factor binding sequence of the PTTG promoter, suggesting that the PTTG gene is a target of p53 and may play a role in the p53-mediated cellular response to DNA damage.
Our results show that overexpression of PTTG activates p53 expression, and that this action of PTTG is achieved through the regulation of the expression of c-myc, which in turn regulates the expression of the p53 gene, by its direct binding to the c-myc/max sequence of the p53 promoter. The importance of c-myc in the induction of the expression of p53 by PTTG is further revealed by our studies using a c-myc dominant-negative construct (Fig 6A and 6B). These results are consistent with those of Kirch et al.  and Levine  who also documented the importance of c-myc for p53 expression and activation. Furthermore, c-myc has been reported to promote apoptosis by destabilizing mitochondrial integrity in cooperation with proapoptotic members of the BCL-2 family including bax  and it is considered as an important transcription factor . Thus, its role in the induction of p53 expression seems to be of physiologic importance in the control of genomic stability in cells.
To assess the significance of the activation of p53 by PTTG with respect to the function of p53 in apoptosis and activation of downstream signaling genes, we analyzed apoptosis using TUNEL staining as well as the activation of the bax promoter. The bax gene encodes a pro-apoptotic member of BCL-2 gene family  and its regulation by p53 is well documented . Our results indicate that overexpression of PTTG induces bax promoter activity (Fig. 7A). PTTG overexpression also induces apoptosis in HEK293 cells (Fig. 8). These results are in agreement with the earlier reports of PTTG overexpression inducing apoptosis by p53-dependent and independent mechanisms . It would seem that when both of these apoptotic pathways fail, PTTG can support the survival of aneuploid cells, thereby supporting tumor growth.
Our studies reveal that the PTTG protein can up-regulate expression of p53 in cells dependent on their p53 status. This stimulatory effect of PTTG is indirect and is mediated through c-myc. PTTG also up-regulates the activity of the bax promoter and increases the expression of bax through modulation of p53 expression.
Construction of reporter plasmids
Sequence of different primers used in PCR amplification Construct
Sense 5'-ATGAGCTC GGATTACTTGCCCTTACT-3'
Antisense 5'-TACTCGAG AATCCAGGGAAGCGTGTC-3'
Sense 5'-ATGAGCTC TTGATGGGATTGGGG TTTTC-3'
Antisense 5'-TACTCGAG AATCCAGGGAAGCGTGTC-3'
Sense 5'-ATGAGCTC CAAAAGTCTAGAGCCACCGT-3'
Antisense 5'-TACTCGAG AATCCAGGGAAGCGTGTC-3'
Sense 5'-ATGAGCTC TTGATGGGATTGGGGTT TTC-3'
Antisense 5'-TACTCGAG ACGGTGGCTCTAGACTTT-3'
Cell culture and transfection
We purchased HEK293, PC-3 and MCF-7 cell lines from American Type Culture Collection (ATCC). All cells were cultured under conditions recommended by the supplier. For transient transfections we seeded the cells in six-well tissue culture plates 24 hours prior to transfection. Cells were transfected with appropriate plasmid DNA (1 μg/well) using Fugene-6 as a transfection reagent (Boehringer Mannheim) as described previously . We used pRenila-Luc (100 ng) (Promega, Madison, Wisconsin) as an internal control. After 48 hours of transfection, the cells were harvested and assayed for luciferase and renilla luciferase activities using dual-luciferase reporter system (Promega) and quantified using a Zylux Femtometer FB12 luminometer.
Western blot analysis
The cells were harvested in lysis buffer (50 mM Tris-HCl, 8.3; 100 mM NaCl; 0.1% Triton X-100; 1 mM PMSF; 1 μg/ml leupeptin; 1μg/ml pepstatin and 1μg/ml aprotinin) and denatured in loading SDS sample buffer by heating at 95°C for 3 minutes. Fifty μg of proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred to Hybond nitrocellulose membrane (Amersham Biosciences). The membranes were blocked in 5% non-fat milk for 1 hour at room temperature, followed by incubation in primary antibody for 1 hour. The antibodies used were anti-PTTG (1:1,500 dilution) , anti-p53 (1:2,000 dilution, Zymed Laboratories, San Francisco, California), anti-bax (1:500 dilution, Sigma, St. Louis, Missouri), and actin (1:5,000 dilution, Sigma). Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 was used to dilute the antibodies and wash the membranes. Proteins were visualized using the ECL system (Amersham Biosciences) according to the supplier's instructions.
Site-directed mutagenesis of the putative c-myc/max binding sequence of the human p53 gene promoter carrying a 3-nucleotides change in p53/-172/-89 construct was carried out using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) using p53/-172/-89 construct as a template and selected primers (sense 5'-TCCCCTCCCTTGGACTCAAGACTGGCGCTA-3' ; antisense 5'-TAGCGCCAGTCT TGAGTCCAAGGGAGGGGA-3') in PCR. Changing the sequence CATCTG in the wild-type sequence to CTTGGA created the mutation in the c-myc/max binding sequence. The construct was sequenced to confirm the mutation and was designated p53/-172/-89-Δ-c-myc.
Preparation of nuclear extracts and DNA footprinting analysis
Nuclear extracts from HEK293 cells transfected with pcDNA3.1 or pcDNA3.1-PTTG were prepared according to Panek et al. . Briefly the cells were collected in ice-cold phosphate-buffered saline (PBS) and pelleted at 3,700 rpm for 10 minutes. The packed cell volume (PCV) of the cells was measured and then the cells were resuspended (3 volumes of PCV) in hypotonic buffer (1 M HEPES, pH 7.9, 1 M MgCl2 and 1 M KCl) and incubated for 10 minutes on ice. The resuspended cells were then transferred to a glass homogenizer and homogenized. The lysis of the cells was confirmed by Trypan blue dye exclusion. After homogenization, the nuclei were sedimented by centrifugation at 4,600 rpm for 15 minutes and then resuspended (3 volumes of the sedimented nuclei) in low-salt buffer (1 M HEPES, pH 7.9, 0.15 M MgCl2, 0.5 M EDTA, 20 mM KCl and 25% glycerol) and mixed gently, followed by addition of high-salt buffer (1 M HEPES, pH 7.9, 0.15 M MgCl2, 0.5 M EDTA, 1 M KCl and 25% glycerol). The high-salt buffer was added drop wise to the mixture with gentle mixing for 30 minutes. The mixture was centrifuged at 5,000 rpm for 15 minutes. The supernatant containing the nuclear extract was dialyzed against 100 volumes of dialysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.2 mM PMSF and 0.5 mM DTT) for 1 hour. The mixture was centrifuged for 20 minutes at 5,000 rpm to pellet any precipitated proteins. and the supernatant containing the nuclear extract was analyzed to determine the protein content and stored at -70°C.
The 84 base pair (-172 to -89) fragment of human p53 promoter was amplified using [γ-32P] end-labeled sense or antisense primer (see Table 1 for primer sequences) and the p53/-172/-89 construct as a template in PCR as described above. This [32P]-labeled DNA fragment was purified on a 2% agarose gel. DNA footprinting analysis was carried out in a 25 μL reaction volume containing 200 mM HEPES, pH 7.9, [32P]-labeled DNA fragment (40,000 cpm) and 50 ng/μl purified PTTG recombinant protein . The binding reaction was allowed to proceed for 10 minutes on ice. The reaction was terminated by addition of 50 μL of stop solution A (10 mM MgCl2, 5 mM CaCl2 and 6.5 ng/μL of yeast transfer RNA) followed by digestion of DNA with 0.2 unit of DNase I (Promega Biotech, WI) for 1 minute at room temperature. The DNA digestion reaction was terminated by addition of stop solution B (20 mM EDTA, pH 8.0, 1.0% SDS and 0.2 M NaCl). DNA was extracted with phenol/chloroform, and separated by electrophoresis through 8% acrylamide/7 M urea gels. The gel was dried and subjected to autoradiography.
Electrophoretic mobility shift assay (EMSA)
The [32P]-labeled DNA probe (84 bp) containing either the wild-type or mutated c-myc/max binding sequence was purified from agarose gel and used in gel shift assays. Briefly, 4 μg of nuclear extract was incubated with the γ-[32P] labeled probe (25,000 cpm) in EMSA buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 250 mM NaCl; 2.5 mM EDTA; 2.5 mM DTT and 20% glycerol) and 250 ng/ml poly (dI-dC) in a 25 μl reaction volume for 30 minutes at room temperature. For super shift assays, the nuclear extract was incubated with PTTG antiserum (1:1,500 diluted), N-terminal anti-c-myc rabbit polyclonal antibody (0.5 μg, Santa Cruz Biotechnology, Santa Cruz, CA) or C-terminal anti-c-myc monoclonal antibody (0.5 μg, Zymed Laboratories, San Francisco, CA) for 30 minutes at room temperature prior to the addition of labeled probe. DNA-protein complexes were separated on 4% polyacrylamide gels and analyzed by autoradiography.
Double immunostaining of cells for PTTG and p53 proteins
HEK293 cells were grown on polylysine-coated chamber slides (Nunc International Corp., Naperville, IL), for 24 hours and then transfected with pcDNA3.1 or pcDNA3.1-PTTG cDNA as described above. After 48 hours of transfection, the cells were fixed with 4% freshly prepared paraformaldehyde for 8 minutes and then treated with 0.1% Nonidet P-40 for 5 minutes. Cells were treated with 5% normal goat serum for 60 minutes to block nonspecific binding followed by incubation with preimmune serum (1:1,500 dilution) plus p53 antibody (1:100 dilution) or PTTG antiserum (1:1,500 dilution)  plus p53 antibody (1:100 dilution) for 60 minutes at room temperature. After several rinses with PBS buffer, the cells were incubated with Alexa Flour® 594 goat anti-mouse and Alexa Flour® 488 goat anti-rabbit secondary antibodies (1:500 dilution, Molecular Probes, Eugene, OR) for 45 minutes. Cells were rinsed with PBS and analyzed using a fluorescent microscope (Olympus X-70).
Apoptosis assay and flow cytometric analysis
Apoptosis analysis of the cells was carried out using TUNEL assay kit (Roche, Indianapolis, IN). HEK293 cells were transiently transfected with pcDNA 3.1-PTTG or p53 plasmid as described above. After 48 hours of transfection, the cells were subjected to TUNEL assay following the supplier's instructions. Briefly, the cells were washed with PBS, air-dried and fixed in 4% paraformaldehyde pH (7.4) for 1 hour at room temperature followed by incubation in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 minutes on ice. The cells were then washed twice with PBS and air-dried. Fifty μl of TUNEL reaction mixture was added to each sample and incubated in the dark in a humidified chamber for 60 minutes at 37°C. The cells were washed three times with PBS and examined under Olympus X-70 fluorescence microscope. For flow cytometric analysis cells were transfected with pcDNA3.1-PTTG or pcDNA3.1-p53 cDNA for 24 hours and analyzed as described by Zhou et al, .
List of abbreviations
- PTTG :
pituitary tumor transforming gene
- Myc DN:
c-myc dominant-negative expression construct
electrophoretic mobility shift assay.
This work was supported by grants from NIH/NCI 82511 and Kentucky Lung Cancer Program.
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