SHORT COMMUNICATION
p53 controls hPar1 function and expression
Z Salah
1, S Haupt
2, M Maoz
1, L Baraz
1, V Rotter
3, T Peretz
1, Y Haupt
2and R Bar-Shavit
11Department of Oncology, Hadassah-University Medical Center, Jerusalem, Israel;2Department of Immunology, Lautenberg Center, The Hebrew University - Hadassah Medical School, Jerusalem, Israel and3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
Human protease-activated receptor 1(hPar1) is a bona fide receptor of the hemostatic protease thrombin, and has a central function in tumor progression. Inactivation of the tumor suppressor gene p53 is one of the most common genomic alterations occurring in cancer. Here, we address the interrelations between p53 and hPar1 in cancer. We demonstrate an inverse correlation between hPar1 gene expression and wild-type (wt) p53 levels, and a direct correlation with levels of the mutant (mt) p53. Bioinfor- matic search revealed the presence of at least two p53 motifs in the hPar1 promoter. Indeed, temperature- sensitive (ts) p53 forms reduced hPar1 promoter activity on wt p53 expression. Ectopic introduction of the p53R175H mutant into cells lacking p53 caused a moderate two-fold induction of hPar1 promoter activity.
Chromatin immunoprecipitation (ChIP) analyses con- firmed a physical association between the p53 protein and hPar1 chromatin fragments. In parallel, PAR1 function is attenuated by p53, as shown by inhibition of pFAK levels and a Matrigel invasion assay. Ectopic reinforcement of hPar1 rescued the inhibition conferred by p53, confirming that p53 directly affects hPar1 expression and function. Altogether, we provide evidence for a direct binding between p53 and hPar1 chromatin, and assign hPar1 as a target of p53.
Oncogene(2008) 27, 6866–6874; doi:10.1038/onc.2008.324;
published online 29 September 2008
Keywords: PAR1; p53; protease; promoter activity;
transcription factor
Introduction
Tumor metastasis is largely dependent on the acquired characteristics of cancer hallmarks, such as limitless replication, ability to escape apoptosis and sustained angiogenesis (Hanahan and Weinberg, 2000). Protease- activated receptor-1, PAR1, the first and prototype member of a PAR family, is emerging as having a
central function in tumor progression. We, and others, have shown pro-angiogenic, pro-survival and pro- invasive properties of PAR1 (Even-Ram et al., 1998;
Chay et al., 2002; Darmoul et al., 2003; Yin et al., 2003;
Caunt et al., 2006; Salah et al., 2007a).
None or very little human protease-activated receptor 1 (hPar1) expression is detected in normal epithelia;
enhanced overexpression is observed in malignant tissues. The mechanism that leads to hPar1 overexpres- sion is as yet unknown. Although impaired internaliza- tion resulting in persistent signaling and invasion was suggested for several breast cancer lines (Booden et al., 2004), an imbalanced expression between hPar1 repres- sors and activators was proposed (Tellez et al., 2003).
While examining the consequences of hPar1 gene overexpression, we have shown that it does not take place on DNA gene amplification or increased mRNA stability. Rather, it is because of increased hPar1 transcription as evaluated by amplified transcription rates in aggressive versus non-aggressive prostate cancer lines. Indeed, we have identified Egr-1 as a critical regulator of hPar1 gene overexpression in the hormone- independent prostate carcinoma (Salah et al., 2005, 2007b).
In the present study, we analysed the interrelations between the p53 tumor suppressor gene and hPar1. We noticed that hPar1 expression is low in cancer lines expressing wild-type (wt) p53, whereas it is over- expressed in cell lines lacking p53 or those expressing mutant (mt) forms. We thus hypothesized that p53 may serve as a potential regulator for the fine-tuning of hPar1 expression during cancer development. The p53 gene encodes a nuclear transcription factor that accumulates in the cell in response to a variety of stress conditions. Under normal conditions, p53 has a short half-life and is thus maintained at low levels. The p53 gene is the most frequent target of genetic alteration yet identified in human cancers, affecting more than 50% of all tumors (Joerger and Fersht, 2007; Soussi, 2007). In most cases, the mutation is located in the DNA-binding core domain of the protein (Olivier et al., 2002;
Hamroun et al., 2006). The functional consequences of p53 cancer mutations are complex (Blagosklonny, 2000;
Sigal and Rotter, 2000; Vousden and Lane, 2007).
Although certain mutants result in p53 loss of function, others exhibit altered transactivation spectra or retain the function at lower temperatures (Kato et al., 2003;
Resnick and Inga, 2003; Dearth et al., 2007).
Received 20 March 2008; revised 21 July 2008; accepted 31 July 2008;
published online 29 September 2008
Correspondence: Dr R Bar-Shavit, Department of Oncology, Hadassah-Hebrew University Hospital, POB 12000, Kiryat Hadassah, Jerusalem 91120, Israel.
E-mail: [email protected]
www.nature.com/onc
Here, we demonstrate the suppressive effect of p53 on hPar1 expression and function. Although an inverse correlation is apparent between wt p53 gene expression and hPar1, mutant p53 forms, representing either loss of p53 or impaired function, directly correlate with hPar1 overexpression. We hereby propose that hPar1 gene is a target of p53.
Results
Inter-relationship betweenhPar1 and p53
We have initiated correlation analyses between p53 and hPar1 expression. For this purpose, we examined different cell lines exhibiting either wt (for example, LNCaP and MCF7) or mt p53 (for example, DU145 and HT29). In parallel, we looked at cancer lines that have lost either p53 expression (for example, PC3 and CL1) or function (for example, WI-38/hTERT/GSE56 cells), whereby p53 activity is downregulated by enforced expression of the genetic suppressor element 56. Genetic suppressor element 56 corresponds to the C-terminal portion of p53, and its overexpression results in the accumulation of p53 in its inactive conformation and the subsequent inhibition of p53 activity (Ossovs- kaya et al., 1996). Our data show that when wt p53 is expressed, hPar1 levels are low or absent (Figures 1a, bi and e). In contrast, when mt p53 is expressed, high hPar1 levels are observed (Figures 1a, d and e).
Collectively, we demonstrated high levels of hPar1 when wtp53 expression (Figures 1a and e) or function is lost (Figure 1bii). To further demonstrate a direct correla- tion between p53 and hPar1, we used H1299 lung adenocarcinoma cells that are p53-negative or stably transfected with a temperature-sensitive (ts) Val135
mutant form of p53 (H1299Val135). This mutant protein contains a substitution of cysteine to valine at position 135, and possesses wt activity at 32 1C and a mutant inactive conformation at 37 1C (Michalovitz et al., 1990). We tested hPar1 mRNA expression in these cells, in which the temperature shift from 37 to 32 1C leads to downregulation of hPar1 mRNA. The levels of p21 in cells expressing ts p53 mutants are indicative of p53 expression and function, as it is hard to detect the very low p53 levels in these cells (Figure 1c). Parental H1299 cells transfected with empty vector show high hPar1 levels with no p21 or p53 expression. When wt p53 was transfected in these cells, a marked reduction in hPar1 levels was observed on a background of elevated p21 and p53 levels (Figure 1c, right panel). To show decisively that the correlation obtained for hPar1 levels stems from an effect of p53 and not another genetic change acquired by these cells, we used p53-silenced MCF7 cells stably expressing p53-SiRNA. In these cells, following silenced p53, hPar1 is now detectable, whereas in parental or empty vector-transfected cells expressing high p53 levels, hPar1 is undetectable (Figure 1bi). This is also shown on PAR1 and p53 protein levels (Figure 1e, right panel). Similarly, when mt p53 expression is lost, hPar1 expression is similarly down-
regulated (Figure 1d). These data suggest a reciprocal correlation between hPar1 and wt p53 expression, and a direct correlation between mt p53 and hPar1.
The effect ofwt and different p53 variants on hPar1 promoter activity
Bioinformatic search for consensus motifs in the hPar1 regulatory region showed two sites at 2936 to 2916 and 1704 to 1724. We then examined hPar1 promoter activity under the different p53 variants. We used H1299Val135 cells expressing p53 ts mutant, co-transfected with the reporter constructs containing a luciferase gene driven by either the hPar1 promoter or the p21 promoter (a known target of p53). Figure 2a illustrates the markedly reduced hPar1 promoter activity in lysates of H1299Val135 cells cultured at 32 1C, as compared with lysates at 37 1C, showing elevated promoter activity. In contrast, the p21 promoter was markedly induced at 32 1C, towing to wt p53 activity (Figure 2b). In the parental p53-negative H1299 cells, both hPar1 and p21 promoter’s activity were unchanged by temperature shift (data not shown), indicating that the observed difference was because of a specific inactivation of ts p53. To further investigate the effect of p53 on hPar1 promoter activity, we introduced hPar1-LUC-promoter plasmid into the p53-negative SaoS-2 cells. These cells were co-transfected with a plasmid expressing either wt p53 or one of its mutant variants (p53-V173L, p53-R175H, or p53H179Q) com- monly found in human cancers. Our data show that the expression of wt p53 in SaoS-2 cells led to a marked decrease in hPar1 promoter activity, reaching a five-fold reduction (Figure 2c). In contrast, none of the three p53 mutants tested displayed inhibitory functions (Figure 2c). Interestingly, the expression of p53- R175H, which is a ‘hot spot’ mutant in human cancers (Vousden and Lu, 2002), caused a moderate (two-fold) but consistently enhanced activity of the hPar1 promo- ter (Figure 2c). Thus, mutant p53 acts to increase the level of the hPar1 gene and may provide part of the molecular regulation of increased hPar1 levels in tumors. The p21-LUC-promoter plasmid was used as a positive control for wt p53 functionality, showing elevated luciferase levels with the wt p53 plasmid (Figure 2d).
In vivo interaction between wt p53 and hPar1:ChIP analysis
To confirm a physical association between the p53 protein and the hPar1 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) analyses. For this purpose, we used a PCR to amplify the relevant chromatin DNA fragments that were specifically im- munocomplex with p53. Chromatin fragments were immunoprecipitated from either cultured WI-38/
hTERT or WI-38/hTERT/GSE56 cells using anti-p53 antibodies and a rabbit IgG serum as control. DNA from the immunoprecipitated complexes was isolated and PCR amplified to include regions between 3046 and 2768 and between 1824 and 1604 bps of the
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proposed p53 sites in the hPar1 promoter. The PCR signal obtained in the non-complexed chromatin (input) was used as a control for DNA loading. When specific
p53 antibodies were used for immunoprecipitation, there was a fivefold increase in the PCR product from WI-38/hTERT cells as compared with WI-38/hTERT/
Figure 1 Correlation between p53 and hPar1 expression: (a–d) RT–PCR and (e) protein analysis. (a) Correlation analyses between hPar1expression and p53. Low levels of hPar1 are observed in cells expressing wt p53 (for example, LNCaP), whereas high hPar1 levels are seen either when mutant (mt) p53 (for example, DU145) is present or when loss of p53 expression takes place (for example, PC3 &
CL1). The levels of p53 in these prostate cancer cells are indicated at the mRNA level. (b) Silencing of p53 rescues hPar1 expression.
Expression of hPar1 is seen when p53 mRNA is downregulated either by p53-SiRNA in MCF7 cells as compared with parental or mock-transfected vector (bi), or when its function is lost by the expression of GSE6 (bii). Although NA silences levels of p53, the functional inhibitor GSE56 does not affect levels of expression (bi, ii and d). (c) Temperature-induced downregulation of p53 in H1299Val135cells result in decreased hPar1 expression. hPar1 level is downregulated in H1299 cells expressing temperature-sensitive (ts) H1299Val135on incubation at 32 1C (cells express wt p53). High levels of hPar1 are observed when cells are incubated at 37 1C (cells exhibit mt, inactive p53 variant). The p21 gene, a direct substrate of p53, is used to demonstrate that the observed differences in gene expression are because of inactivation of ts p53 rather than to a general effect of the temperature change. The levels of hPar1 are high in the parental mock-transfected H1299 cells (H1299 empty vec) showing no p21 or p53 expression. H1299 cells transfected with wt p53 show reduced hPar1 levels and elevated p21 and p53 levels. (d) SiRNA knockdown of mt p53 levels reduces hPar1 expression levels.
High levels of hPar1 expression are observed in either the mt p53 (for example, HT29 cells) or empty vector-transfected HT29 cells (empty vec). In contrast, hPar1 levels are potently inhibited when p53-SiRNA potently silences p53 levels, leading to downregulation of hPar1expression. (e) Protein levels of hPar1 and p53: western blot analysis. PAR1 levels are high in cells expressing mt p53 (for example, Du145) or in cells exhibiting loss of wt p53 (for example, PC3 and CL1) and low in cells expressing wt p53 (for example, LNCaP, MCF7 empty vector transfected (empty vec) and MCF7 (data not shown)). Induced levels of PAR1 protein are observed in cells exhibiting p53 -SiRNA silenced p53 level. The above protein levels are obtained following equal loading as indicated by b-actin levels. These data are representative of at least three experiments performed for each cell line.
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GSE56 cells in which p53 is functionally inactive. When control rabbit IgG was used to immunoprecipitate the chromatin, only a minimal amount of PCR product was obtained, representing a non-specific residual product.
In control IgG samples, there was no difference in the amount of PCR product generated from immunopreci- pitated chromatin regardless of the cells used (Figure 3).
To show that there is no binding site of p53 in the 30or 50UTR other than the identified binding sites, we performed PCR using primers located either in the 30UTR or in the 50UTR but not in the vicinity of the identified p53-binding sites, showing no product gen- eration (data not shown). These results demonstrate that p53 physically associates with the hPar1 promoter in vivo.
p53 attenuates PAR1 functionality
We tested whether the PAR1-signaling pathway is affected by either wt and/or mt p53 expression. For this purpose, we examined the phosphorylation status of a known PAR1 target protein, focal adhesion kinase
(FAK). FAK has been shown to undergo phosphoryla- tion on PAR1 activation (Even-Ram et al., 2001). When we incubated H1299Val135 cells at 32 1C or 37 1C and analysed the levels of pFAK following activation with either SFLLRN, a PAR1 ligand peptide, or thrombin as a ligand (data not shown), the following data were obtained. Although nearly no change in pFAK levels was seen at 32 1C (wt p53 is expressed), a marked increase was observed in cells at 37 1C (mt p53 is expressed) (Figure 4a). Indeed, cells incubated at 32 1C showed very little to no PAR1 protein levels. In contrast, equal PAR1 protein levels are observed at 37 1C (Figure 4a). Induced pFAK at 37 1C as compared with 32 1C is represented by histograms at the right panel of the figure (Figure 4a). To further show that PAR1 functionality is attenuated, we performed a Matrigel invasion assay using H1299Val135cells. PAR1 activated cells show a significant inhibition in invasion as observed on temperature shift from 37 to 32 1C, indicative of the inhibitory impact of wt p53, versus the inactive mutant p53 on PAR1 invasive function.
Impaired invasion did not result from cell viability at
Figure 2 (a) Temperature-induced inactivation of H1299Val135variant results in decreased hPar1 promoter activity. H1299Val135
cells were transiently transfected with the LUC reporter gene driven by either the hPar1 (a) or the p21 (b) promoter. Immediately following transfection, the cells were incubated at 32 or 37 1C for 48 h, lysed and measured for luciferase activity. The relative light units in each sample (±s.d.) were normalized against b-galactosidase activity measured by a colorimetric assay. A marked decrease in hPar1 promotor activity was observed when cells were incubated at 32 1C (a) as compared with increased p21 promoter activity.
(b) Effect of different p53 variants on hPar1 promoter activity. SaoS-2 cells were co-transfected with the luciferase reporter gene driven by either the hPar1 (a) or the p21 (b) promoter and either empty vector (Vec) or vectors encoding for wt or one of the three mutated variants (p53-V173L, p53-R175H and p53-H179Q) of p53. Equal amounts of DNA in each well were maintained by the addition of an empty vector. Luciferase light units and b-galactosidase colorimetric assay activities were measured 24 h later. The graph represents fold difference in the promoter activity under different conditions. These data are representative of at least four experiments performed.
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32 1C, as a high number of viable cells was found on the other side of the membrane (data not shown). To demonstrate that this phenomenon correlated with attenuated hPar1 gene expression and not with other p53 target genes, we re-expressed hPar1 ectopically by retroviral infection. In these cells, the impaired invasion was clearly rescued (Figure 4b). Levels of hPar1 expression were verified by RT–PCR after infection, further demonstrating that the induction in hPar1 expression is not affected by a temperature shift (Figure 1c). Matrigel invasion assay performed in the presence of a potent PAR1 antagonist SCH 79797 (Tocris Cookson Inc., Ellisville, MO USA) showed a marked inhibition in the invasion. Inhibition was obtained under the various treatments applied and the ectopic application of hPar1 to H1299 p53 cells at either 37 or 32 1C. This shows that the invasion is a direct result of activated PAR1. Histograms appearing at the right panel represent levels of invasion under different treatment conditions (Figure 4b).
Discussion
In this study, we investigated the role of p53 in the regulation of hPar1 expression and function. For this purpose, we used molecular probes of p53, including ts- and function-blocking mutants, luciferase promoter constructs and chromatin-protein analyses. We found that the hPar1 levels correlated inversely with wt p53 expression and directly with mt p53 expression.
Analysis of the molecular mechanism underlying this interaction indicated that wt p53 inhibits hPar1 expres- sion by direct binding to consensus p53 sites, as shown by the hPar1 luciferase promoter activity and ChIP analyses.
The promoter sequence of thrombin receptor/PAR1 was cloned simultaneously by two groups (Li et al., 1996; Schmidt et al., 1996). It was proposed (Li et al., 1996) that the 50 regulatory region of PAR1 contains consensus-binding sites for numerous regulatory pro- teins. The identification of discrete proteins that bind to
Figure 3 Chromatin immunoprecipitation: ChIP demonstrates the in vivo interaction between p53 and the hPar1 motifs for p53.
(a) The hPar1 promoter sequence, highlighting p53 consensus sites and the primers used for ChIP analyses. (b) Upper panel: DNA fragments immunoprecipitated with the indicated antibodies were purified, and the region containing p53 motifs was amplified by PCR. An equal amount (input) of DNA-protein complex was applied. PCR products generated using hPar1 promoter primers or GAPDH primers to amplify immunoprecipitated DNA of cells expressing either non-functional p53 protein (WI-38/hTERT/GSE56, lane 1) or functional p53 protein (WI-38/hTERT, lane 2) are shown. Non-specific low levels were obtained when a non-specific control IgG was used to immunoprecipitate the complex (not seen when amplified using the same number of cycles). The p21 promoter primers were used as a positive control for the experiment. This is a representative experiment of at least three independent trials.
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the PAR1 promoter is starting to emerge. Among the activators are Sp1 (Tellez et al., 2003), androgen/
hormone response elements (Salah et al., 2005) and Egr-1 (Salah et al., 2007a). The repressors are AP-2 (Tellez et al., 2003) and wt p53 (this study).
p53 binds the p53 consensus sites to repress promoter activity either directly (Laptenko and Prives, 2006;
Levine et al., 2006) or indirectly through binding to the TATA-binding protein (Farmer et al., 1996) or other
transcriptional factors. Bioinformatic search for the p53 consensus sequence (el-Deiry et al., 1992) revealed two motifs, in the hPar1 promoter residing between 2936 to 2916 and 1704 to 1724, upstream to the PAR1 coding region. Although the first site matches the traditional p53-binding consensus motif with one mis- match, the second site matches only half of the p53- binding consensus motif (Weinberg et al., 2005;
Menendez et al., 2006).
Figure 4 p53 attenuates PAR1 function: PAR1-induced pFAK and invasion. (a) Levels of pFAK. H1299Val135cells were incubated at either 32 or 37 1C and activated by the PAR1 ligand peptide (SFLLRN) for the indicated time periods. Although there is no increase in pFAK levels above control in cells incubated at 32 1C (wt p53 is expressed), a marked increase is observed in cells incubated at 37 1C (mt p53 is expressed). The levels of induced pFAK by PAR1 activation are obtained on a background of equal protein levels of FAK.
PAR1 protein levels are markedly inhibited when wt p53 is expressed at 32 1C as compared to equal PAR1 levels at 37 1C (mt p53 is expressed). Representative quantitative demonstration of the result is shown in the histogram, right panel a, after normalizing results against FAK protein levels. These data are representative of at least three experiments performed. (b) Matrigel invasion assay.
A marked inhibition of invasion is observed when cells are incubated at 32 1C (H1299 p53/321C) as compared with cells incubated at 37 1C (H1299 p53/371C). Ectopic re-expression of hPar1 rescued the inhibitory effect induced by the induction of wt p53 expression (H1299 p53/Par1/32 1C), but it had no effect on cells incubated at 37 1C (H1299 p53/371C). No effect is observed with temperature shift in H1299 naive cells (data not shown). All treatments were performed on either thrombin-activated PAR1 cells (þ ) or SFLLRN- activated (data not shown) as compared to non-treated control cells (). When the invasion assay was performed in the presence of PAR1 antagonist (SCH 79797, Tocris Cookson Inc., Ellisville, MO, USA), a marked inhibition throughout the various treatments was obtained, indicating that the invasion is a direct outcome of a functionally active PAR1. Quantitative presentation of the invasion data is shown by histograms±s.d., right panel b. Activated PAR1 in H1299 p53/37 1C shows 85–91 cells per field and the control non- activated cells show 8–12 cells per field. These data are representative of at least five experiments performed.
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The ChIP analyses decisively demonstrate a direct binding of p53 to the hPar1 promoter. p53 also appears capable of trans-repressing genes that participate in various signaling pathways such as cell proliferation (cyclin B, RNA polymerase I and c-myc), apoptosis (bcl-2 and survivin) and cytoskeletal organization (stathmin and map4) (Ahn et al., 1999; Ho and Benchimol, 2003). Here, we demonstrated a p53 trans- repressing effect on PAR-1 expression. We further demonstrated the consequences of the p53 inhibitory effects on PAR1 functionality, as shown by the inhibition of PAR1-induced levels of pFAK and invasion capability, alleviated by the ectopic re-expres- sion of hPar1.
Malignant tumors disrupt the p53-signaling pathway to grow and survive (Vousden and Lu, 2002; Martins et al., 2006) Although many genes in addition to p53 are mutated in tumors, the restoration of the p53 function alone is sufficient to cause regression of several types of tumors (Martins et al., 2006; Kastan, 2007; Sharpless and DePinho, 2007). Unlike other tumor suppressor genes, p53 is inactivated by missense mutations in approximately half of all human cancers (Strano et al., 2007). In fact, the expression of p53 mutants tend to support oncogenic functions favoring the insurgence, maintenance, spreading and chemoresistant properties of tumors (Sigal and Rotter, 2000). In the present study, we provided evidence to support the notion that in addition to wt p53, mt p53 is implicated in the regulation of hPar1 expression. We showed that in different cell lines (for example, DU-145 and HT-29), exhibiting various p53 mutant forms, hPar1 is highly expressed, and that when mt p53-SiRNA is applied, hPar1 expression is downregulated. Mutant p53 acts through different mechanisms, including gain of transcriptional activity (Prowald et al., 2007). In the present study, we demonstrated that at least one p53 variant (p53-R175H) is capable of inducing hPar1 promoter activity. Whether this enhanced transcription occurs by binding of mt p53 to a specific motif in the hPar1 promoter, or indirectly through complex formation with other DNA-binding factors, needs to be elucidated. Mutant p53 (p53- R175H) induces Egr-1 by physical association with the Egr-1promoter providing a significant gain of function (Weisz et al., 2004). Egr-1 targets the hPar1 promoter in prostate cancer, leading to its overexpression and induction of invasive properties (Salah et al., 2007a).
Altogether, hPar1 expression may be induced by mt p53 through elicited Egr-1 expression, a hypothesis that needs to be determined.
Mutants p53 act either through a dominant-negative function or a gain-of-function (Sigal and Rotter, 2000;
Cadwell and Zambetti, 2001). Dominant-negative comes from observations that mutant p53 oligomerizes with wt p53 and inhibits its function (Milner et al., 1991; Willis et al., 2004). In contrast, p53 mutants expressed in tumors exhibit gain-of-function properties, contributing to the overexpression of hPar1 (because ectopic application of p53 -SiRNA in HT-29 colon cancer cells possessing mt p53 His273, reduced markedly high hPar1 levels). Another mode of mt p53 gain of function is
observed in prostate cancer cells. Although DU-145 of high hPar1 express mt P223L p53, in PC3 also of elicited hPar1 levels, p53 is lost as a result of a frame shift deletion at codon138 (Isaacs et al., 1991). How these mutants interact with their target promoters is still unclear. Although wt p53 is a sequence-specific trans- cription factor negatively regulated by Mdm2, an E3 ubiquitin ligase targeting p53 for ubiquitination and proteasomal degradation, the consensus-binding sites of mutant p53 are unknown. One possibility is the binding through NF-Y known to bind to the CCAAT motif (Di Agostino et al., 2006), two of which are found within the hPar1 promoter (at2736 and 2516 ) to possibly recruit mt p53. In the presence of mt p53, the acetylase p300 is preferentially bound to the complex locally assisting in the chromatin to ‘open’ the gene for transcription (Aylon and Oren, 2007).
PAR1 mRNA can be inhibited through enhanced levels of cAMP or induced protein kinase C (PKC).
(Nystedt et al., 1996) . The physiological relevance of this type of regulation remains yet to be fully determined.
PAR1 has also been shown to downregulate p53 (Riewald and Ruf, 2005). Indeed, several transcription factors that act on PAR1 as a target are also regulated by PAR1. This feedback loop has been shown for EGR- 1 (Wu et al., 2002; Salah et al., 2007a) and Groa (Caunt et al., 2006). PAR1 inhibition of p53 can be explained indirectly by PAR1-induced activation of PI3 kinase/
Akt signaling necessary for nuclear entry of Mdm2 (Mayo and Donner, 2001) and p53 degradation.
Activation of PI3K /Akt results in the phosphorylation of Mdm2 acquired for the translocation of Mdm2 to the nucleus. Overall, PAR1, a known survival factor (Salah et al., 2007b) induces the pro-survival signaling pathway and activates PI3K and Mdm2 entry into the nucleus, reducing p53 expression and activity. This fits well with our observation whereby when high levels of PAR1 are observed; p53 levels are very low or lost.
Our observations suggest that the suppression of hPar1 expression by wt p53 may be an important mechanism by which wt p53 suppresses tumor progres- sion in human carcinomas and that the induction of hPar1 expression by mt p53 may be an additional important mechanism by which mt p53 induces tumor progression.
Materials and methods Cell lines
Human prostate adenocarcinoma lines LNCap, CL1, PC3 and DU-145, osteosarcoma SaoS-2, breast carcinoma MCF-7 and lung carcinoma H1299 were cultured in RPMI-1640(Chen et al., 1996; Even-Ram et al., 1998; Salah et al., 2007a). HT29 and H1299 Val135cells were cultured in DMEM (Michalovitz et al., 1990). Primary human embryonic lung fibroblasts (WI- 38), expressed hTERT alone or hTERT and GSE56 as described earlier (Ossovskaya et al., 1996).
RNA extraction and RT–PCR
RNA and RT–-PCR as earlier (Salah et al., 2007a).
p21 sense: 50-ATGTCAGAACCGGCTGGGGA-30 6872
p21 antisense 50-GCCGTTTTCGACCCTGAGAG-30 p53 sense: 50-GTCGACCCCCCTCTGAGTCAGG-30 p53 antisense: 50-GCTGGTGCAGGGGCCACGCG-30. Transfection and luciferase (LUC) activity assay
LUC activity in SaoS-2 as earlier (Salah et al., 2005, 2007a).
The reporter construct (300 ng per well) of the hPar1 promoter HTR/-4.1(F1) or p21-LUC was mixed with wt or p53 mutant constructs (300 ng per well; p53-V173L, p53-R175H and p53H179Q) (kindly provided by Dr K Vousden, The Beatson Institute for Cancer Research UK and Dr M Oren, Weizmann Institute of Science, Rehovot, Israel). H1299Val135 cells were transfected with Poluc- or p21-LUC. All results were normali- zed against b-galactosidase activity measured by a colorimetric assay.
Matrigel invasion assay
Transwell Boiden chemotaxis chambers and 13 mm diameter filters were used for this assay. Polyvinylpyrrolidone-free polycarbonate filters, 8 mm pore size (Costar Scientific Co., Cambridge, MA, USA), were coated with basement membrane Matrigel (200 ng/ml g/filter), as described earlier (Albini et al., 1987). Briefly, cells (2 105) were applied to the upper chamber compartment. Conditioned medium of 3T3 fibroblasts was used as a chemoattractant and placed in the lower compart- ment. At the end of the incubation period, the cells on the upper surface of the filter were removed by wiping with a cotton swab. Filters were fixed, stained with DifQuick System and counted to determine the number of cells at the lower surface of the filter.
Western blotting analysis
Lysis of cells and western blot as earlier (Salah et al., 2007a).
Chromatin immunoprecipitation and PCR analysis
WI-38/hTERT and WI-38/hTERT/GSE56 cells were fixed to cross-link histone proteins to DNA by formaldehyde added to the culture medium (1%, 10min, 25 1C). Glycine (0.125M) was then added to plates to quench formaldehyde. Soluble chromatin was prepared and sonicated as previously described (Salah et al., 2005, 2007a). For immunoprecipitation, anti-p53 antibodies (10 mg), pre-bound to protein A, were added to 500 ml of the purified chromatin sample and incubated over- night at 4 1C. PCR primer sets were designed to overlap the p53-binding sites as follows:
Primer2 sense 50-ctatgttaggaatataaatg-30, antisense 50-ctggg caacagagcaagact-30 Primer4 sense 50-gtgaaattagccggacatgg-30, antisense 50-ctgcgcatctgggaggagg-30. Other primer sets were used to ensure that there were no other p53-binding sites in either the 50- or 30-UTR. Quantitative PCR was performed on the eluted p53 immunocomplexed DNA using the TITANIUM Taq PCR kit (CLONTECH Laboratories Inc., CA, USA).
Acknowledgements
Grant support: Israel Science Foundation by the Israel Academy of Science and Humanities, The Israel Cancer Association and MECC (Middle East Cancer Consortium)—
Small Grant Program (R Bar-Shavit).
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