Introduction

The Wnt/-catenin signalling pathway participates in controlling cell growth. It leads to the activation of gene expression by the T-cell factor (Tcf) and lymphoid enhancer factor (Lef) protein families, which in turn regulate transcription from growth-related genes (Hülsken and Behrens, 2002). Tcf-4 is a main transcription factor to pass on Wnt/-catenin signalling (Korinek et al., 1997).

Wnt proteins are secreted molecules that regulate proliferation and differentiation through interaction with the Frizzled family of cell-surface receptors with seven transmembrane loops (Taipale and Beachy, 2001). Wnt/Frizzled signalling leads to the stabilization of cytosolic -catenin. In the absence of such a signal, glycogen synthase kinase-3 phosphorylates -catenin, which leads to the ubiquitination of this protein and to its degradation via the 26S proteasome (Maniatis, 1999). However, when -catenin is stabilized, it can translocate to the nucleus and serve as a coactivator for Tcf/Lef transcription factors (Behrens et al., 1996; Molenaar et al., 1996; Eastman and Grosschedl, 1999). One activity, counteracting this nuclear transport, originates from the adenomatous polyposis coli (APC) tumour suppressor protein, which regulates the export of -catenin from the nucleus to the cytosol (Rosin-Arbesfeld et al., 2003). Examples for growth- and metastasis-related target genes of the Tcf family are urokinase-plasminogen activator receptor (uPAR), c-Myc and cyclin D1 (Mann et al., 1999; Hülsken and Behrens, 2002). Many components of the -catenin/TCF regulatory network are proto-oncogenes or tumour suppressors, and have thus been directly implicated in the development of cancer. Especially, Tcf-4 has been shown to be relevant for tumour formation. Tcf-4, constitutively activated by mutated -catenin, was identified as an early event in the development of colon carcinoma (Korinek et al., 1997; Roose and Clevers, 1999). Recently, the -catenin/TCF-4 complex has been described as a master switch controlling proliferation versus differentiation in intestinal epithelial cells. In these experiments, a dominant-negative form of Tcf-4, which can no longer be activated by -catenin, induced G₁ arrest and concomitantly led to the initiation of a differentiation programme. The Tcf-4 target gene c-Myc plays a central role in this switch by direct repression of the p21^WAF1/CIP1 promoter. Following disruption of -catenin/Tcf-4-activity, the decreased expression of c-Myc will release p21^WAF1/CIP1 transcription, which in turn mediates G₁ arrest and differentiation (van de Wetering et al., 2002).

The p53 protein is an important tumour suppressor that is inactivated in most tumours. It functions as a transcription factor on target genes to regulate cell-cycle checkpoints and apoptosis (el-Deiry et al., 1993; Ko and Prives, 1996; Contente et al., 2002). p53 is connected to several cellular signalling pathways and serves as an integrator of numerous signals affecting cell growth and apoptosis. Many apoptotic signals are funnelled through p53 to the apoptotic machinery (Vogelstein et al., 2000). Resistance to undergo programmed cell death in human cancers is often associated with loss of p53 function through mutation of its gene (Hanahan and Weinberg, 2000).

p53 exerts its functions mainly as a transcription factor. One example of the many genes that p53 transcriptionally regulates is bax (Miyashita and Reed, 1995). Induction of bax expression produces a protein that accelerates programmed cell death by counteracting the function of Bcl-2 (Reed, 1994). Another prominent target for p53-dependent transcriptional activation is the cyclin-dependent kinase inhibitor p21^WAF1/CIP1 (el-Deiry et al., 1993). p53 is thought to exert its function in G₁ checkpoint control mainly through increased expression from the p21 gene (Brugarolas et al., 1995; Deng et al., 1995). However, a function of p21 and p53 also in G₂ checkpoint control was observed. It has been shown that in colorectal cancer cell lines, p53 and p21 are necessary to maintain a G₂ arrest after -irradiation (Bunz et al., 1998). Other ways by which p53 controls the G₂/M checkpoint is through regulators like 14-3-3 (Hermeking et al., 1997), GADD45 (Wang et al., 1999) and cdc2 (Yun et al., 1999). We have described that transcriptional downregulation of cdc25C (Krause et al., 2001; Manni et al., 2001), cyclin B1 and cyclin B2 (Krause et al., 2000) contributes to p53's function in regulating G₂ arrest and thereby controls entry into mitosis. Besides transcriptional regulation, there have only been a limited number of descriptions on other p53 functions, for example, complex formation (Bates and Vousden, 1999). One other such function is the direct role of p53 in DNA repair, which in part stems from its exonuclease activity (Mummenbrauer et al., 1996; Albrechtsen et al., 1999). Taken together, the main contribution of p53 in growth arrest and apoptosis control appears to originate from its transcriptional activity, by both activation and repression of target genes. Recently, it has been described, in a system using mouse splenocytes, that the p53-dependent response can depend on the proliferation status of the cells. When resting cells were -irradiated they were driven into p53-dependent apoptosis, whereas growth-stimulated cells went into growth arrest after irradiation (Heinrichs and Deppert, 2003). Interestingly, the different responses were independent of the p53-dependent transcriptional programme, at least for the genes selected for testing.

Since p53 exerts most of its functions by transcriptional control, we were interested in identifying new transcriptional targets of p53 by an unbiased approach employing DNA microarray hybridizations. Here, we show that transcription of Tcf-4 is downregulated by p53. This signalling pathway directly connects p53-dependent growth regulation with Tcf/Lef transcriptional target gene control.

Results

Expression of Tcf-4 mRNA is downregulated after increased p53 expression

The human colorectal adenocarcinoma cell line DLD-1 is functionally negative for p53. With a wild-type p53 construct stably introduced into these cells p53 function can be restored, regulated by a tet-off system (Gossen and Bujard, 1992; Yu et al., 1999). In control experiments with this cell system, we confirmed that after tet-off induction expression of p53 mRNA and protein increase (Krause et al., 2000). We employed the DLD-1-tet-off-p53 cells to search for yet unrecognized p53 target genes. In an indicator DNA microarray hybridization experiment using RNA from these cells without and 9 h after the induction of p53 expression, we found Tcf-4 mRNA to be 14.6-fold downregulated when wild-type p53 was present relative to the uninduced state (Table 1). In this experiment also the regulation of known p53 target genes was monitored as control. Consistent with previous observations in other experimental systems, mRNA levels for the cyclin-dependent kinase inhibitor p21^WAF1/CIP1 (el-Deiry et al., 1993), mdm2 (Barak et al., 1993) and bbc3/PUMA (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001) were found to be upregulated when wild-type p53 was expressed in DLD-1 cells (Table 1). As another control, the previously described downregulation of cyclin A (Desdouets et al., 1996), cyclin B (Innocente et al., 1999; Krause et al., 2000), cdc2 (Yun et al., 1999) and cdc25C (Krause et al., 2001; Manni et al., 2001) mRNA levels was also observed in our microarray experiment (Table 1). The p53-inducible DLD-1 cell system had been employed earlier to monitor changes in expression after 9 h of p53 induction. Importantly, it had been shown that under these experimental conditions changes in expression precede the changes due to impending apoptosis (Yu et al., 1999; Krause et al., 2000). The result that p53 increase correlated with a reduction in Tcf-4 mRNA levels found in the microarray analysis served just as an indicator. We confirmed p53-dependent decrease of Tcf-4 mRNA levels by real-time RT–PCR quantification in two independent pairs of DLD-1-tet-off-p53 RNA samples (Table 1). The experiments yielded an average 4.65-fold downregulation of Tcf-4 mRNA from similarly treated cells as in the microarray experiment. The GAPDH mRNA level, which is considered to remain unchanged during p53 induction, served as a standard for Tcf-4 mRNA quantification by RT–PCR. The different methods of signal measurement and standardization associated with these techniques may yield the varying numbers by which Tcf-4 mRNA levels drop upon an increase in p53 (Table 1). Combining results from microarray and RT–PCR analyses, it appeared that Tcf-4 mRNA levels are downregulated when p53 concentration increases. This downregulation preceded signs of p53-dependent apoptosis manifestation.

Table 1 - Change of mRNA levels after induced p53 expression detected by DNA microarray hybridization or RT–PCR.

Full table

p53 downregulates transcription from the Tcf-4 promoter in a dose-dependent manner and in different cell types

In order to elucidate whether this decrease in Tcf-4 mRNA is dependent on the function of p53 as a regulator of transcription, we examined the regulation of the Tcf-4 promoter. To this end, we isolated the Tcf-4 promoter by amplifying human DNA upstream of the Tcf-4 coding region in several steps from an adaptor-ligated genomic DNA library starting with nested gene-specific primers derived from the 5'-end of the cDNA. We created a reporter construct by cloning a 1308 base pair fragment upstream of the translational start codon into a vector to derive the expression of a firefly luciferase reporter (Tcf-4-Luci).

The Tcf-4-Luci plasmid was cotransfected in C33A cervical carcinoma cells with expression constructs for wild-type or mutant p53. Increasing amounts of wild-type p53 led to a downregulation of the Tcf-4 reporter. To control for transfection efficiencies, pRL-null plasmid expressing Renilla luciferase independent of a specific promoter were cotransfected. Furthermore, the expression of a mutant p53 control at the highest concentration had only a small effect (Figure 1a). It had been reported earlier that after p53 expression, under the same experimental conditions as employed here, serving as a positive control, transcription from the p21^WAF1/CIP1 promoter was stimulated and cells were not yet apoptotic (Krause et al., 2001,2000). Cotransfection experiments with the Tcf-4 reporter plasmid and p53 expression vectors were then performed in human colorectal adenocarcinoma cells DLD-1, human foreskin fibroblasts HFF, human osteosarcoma cells SaOS-2, human cervical carcinoma cells C33A and mouse fibroblasts NIH3T3. Wild-type p53 downregulates transcription from the Tcf-4 promoter in all cell systems (Figure 1b). A small reduction of reporter activity is already seen with the p53 mutant. This may be due to transcriptionally relevant interactions by parts of p53mut outside the inactivated DNA-binding site. However, in all cells tested, a substantial wild-type p53-dependent downregulation of the Tcf-4 promoter was observed, albeit at a varying degree (Figure 1b). In summary, this suggests that transcription from the Tcf-4 promoter is negatively regulated through p53-dependent transcriptional activity.

Figure 1.

p53 downregulates transcription from the Tcf-4 promoter. Either a plasmid coding for wild-type p53 or a DNA-binding mutant of p53 or pUC19 vector as a control was cotransfected along with the Tcf-4 promoter reporter and a transfection control plasmid expressing Renilla luciferase. Firefly luciferase activity relative to the Renilla control is given. Experiments were carried out in triplicate with standard deviations indicated. (a) The Tcf-4-Luci construct carrying the human Tcf-4 promoter was cotransfected in C33A cells with increasing amounts of a wild-type p53 expressing plasmid or a plasmid carrying a mutant form of p53. (b) Different cell lines were transfected with the Tcf-4-promoter reporter Tcf-4-Luci together with the wild-type or mutant p53-expressing plasmids or pUC19 as a vector control. Maximum expression for each cell line was adjusted to 100%

Full figure and legend (106K)

Tcf-4 protein levels are also reduced after p53 induction

Western blot analysis of nuclear extracts from DLD-1-tet-off-p53 cells without and after induced expression of wild-type p53 led to a moderate reduction of Tcf-4 protein expression already 9 h after p53 induction (Figure 2a). We had observed earlier that after this time period neither a significant number of cells have become apoptotic nor a strong shift in cell-cycle distribution of these cells due to cell-cycle arrest occurs (Krause et al., 2000). Consistently, another of our controls showed that only a moderate increase in caspase activity is measured after 9 h of wild-type p53 induction in these cells (Dietz et al., 2002). Furthermore, the induction of endogenous p53 expression by ionizing radiation led to a small reduction of Tcf-4 protein in cells that contain wild-type p53. The same cell line, but with a deleted p53 gene, did not show such a downregulation (Figure 2b). These observations suggest that a reduction of Tcf-4 levels dependent on p53 induction can also occur in a normal cell setting.

Figure 2.

Western blot analysis of Tcf-4 expression after the induction of p53. Nuclear extracts were prepared from the different cell lines and analysed with antibodies directed against Tcf-4, p53 and -actin. (a) DLD-1-tet-off-p53 cells without (-) and with (+) induction of wild-type p53 expression for 9 h. Protein (60 g) was used for each lane. (b) Parental HCT116 cells and HCT116 cells with targeted deletion in both p53 alleles without irradiation (control) and after treatment with X-rays (5 Gy). Protein (200 g) was used for each lane

Full figure and legend (84K)

Tcf-4 target gene expression is downregulated after increased p53 expression

Since Tcf-4 itself is a transcription factor, a reduction of expression of its target genes would be expected once Tcf-4 protein levels diminish. The uPAR gene is a target for Tcf/Lef activation (Mann et al., 1999). We tested uPAR mRNA expression 9 h after the induction of p53 in DLD-1-tet-off-p53 cells and found it 5.3-fold reduced in comparison with the uninduced state with GAPDH as the expression standard (Figure 3). However, since these Tcf-4 target genes may also be regulated by other Tcf/Lef family members with overlapping DNA-binding specificity (Roose and Clevers, 1999), this result can only serve as an indicator for Tcf-4 downstream regulation.

Figure 3.

mRNA of the TCF target gene uPAR is downregulated by p53. DLD-1 colorectal cells with a tet-off-regulated gene for wild-type or a DNA-binding mutant of p53 were induced to express p53 wild-type or mutant p53 protein. Relative mRNA levels for uPAR were measured by real-time RT–PCR with and without induction. mRNA levels from cells without induction of p53 protein was set as the 100% reference. Analyses from three independent experiments with standard deviations are shown

Full figure and legend (49K)

Timing of Tcf-4 downregulation follows p53 induction and is dependent on wild-type p53

In DLD-1-tet-off-p53 cells, p53 expression was induced and mRNA expression of p53 and Tcf-4 was followed at several time points up to 9 h after induction by real-time RT–PCR quantification. p53 mRNA levels started to rise at about 40 min after antibiotic withdrawal and continued to increase up to the end of the observation period at 9 h. In contrast, Tcf-4 mRNA levels first remained unchanged and only started to drop after 2 h (Figure 4a), suggesting that Tcf-4 is an effector of p53. Downregulation of Tcf-4 mRNA is dependent on wild-type p53, since induction of a mutant p53 protein did not yield a reduction of Tcf-4 mRNA (Figure 4b).

Figure 4.

Timing of Tcf-4 mRNA expression follows that of p53 mRNA and is dependent on wild-type p53. (a) Expression of mRNA was monitored by real-time RT–PCR in samples from DLD-1-tet-off-p53 cells at different time points without and after the induction of p53 expression. (b) Tcf-4 mRNA expression was compared in wild-type and mutant p53-expressing cells. Analyses from three independent experiments with standard deviations are shown

Full figure and legend (110K)

Downregulation of Tcf-4 transcription is obviously independent of p53 binding to its promoter

Transcriptional activation by p53 is generally associated with p53 binding to the regulated promoter. Two p53-binding motifs had been identified (el-Deiry et al., 1992; Contente et al., 2002). In case of transcriptional repression, direct binding of p53 to a consensus site in the downregulated promoter is usually not detected. We inspected the Tcf-4 promoter (GenBank accession number AF522996) for potential p53-binding sites. Neither a microsatellite with multiple 5'-TG(C/T)CC-3' sequences nor two copies of the 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' motif were found. Also, like other genes repressed by p53, Tcf-4 does not contain a TATA-box in its promoter.

In addition to scanning the promoter for potential binding sites, we carried out chromatin immunoprecipitations (ChIPs) of the Tcf-4 promoter to test for p53 binding to the promoter. With an antibody directed against p53, no Tcf-4 promoter DNA could be co-precipitated and subsequently PCR amplified. The p21^WAF1/CIP1 promoter served as a positive control for p53 binding (Figure 5). Taken together, sequence and ChIP analyses indicate that p53 does not bind to the Tcf-4 promoter as observed for other p53-repressed genes.

Figure 5.

ChIPs with Tcf-4 and p21^WAF1/CIP1 promoters without and after induced p53 expression. Nuclear extracts of DLD-1-tet-off-p53 cells without (-) and after (+) induction of wild-type p53 expression for 9 h were analysed. The p21WAF1/CIP1 promoter that has been shown to bind p53 directly served as positive control. Differences in band mobility for the same DNA fragments in different lanes are due to a slower migration of smaller DNA amounts since SYBR Green was used as a staining dye

Full figure and legend (67K)

p53-dependent downregulation of a Tcf-responsive promoter even in the presence of a stable -catenin mutant

p53 and -catenin proteins are connected by an autoregulatory loop and thereby may affect the Tcf-4-dependent transcriptional response (Damalas et al., 1999; Matsuzawa and Reed, 2001; Sadot et al., 2001). Therefore, we investigated whether p53 could downregulate Tcf-4 target genes independent of this autoregulation, which modulates -catenin concentration. To this end, the pTOPFLASH reporter plasmid, which drives luciferase expression from binding sites for Tcf/Lef transcription factors, was employed. The activity of this Tcf/Lef-responsive reporter was tested in the presence of the degradation-resistant -catenin mutant S33Y (Figure 6). The influence of wild-type and mutant p53 was compared. When no exogenous -catenin is expressed in C33A cells reporter activity is low. However, in the presence of a degradation-resistant -catenin mutant that continuously activates Tcf transcription factors, pTOPFLASH-reporter activity increases drastically. Even in the presence of constitutively active -catenin, p53 was able to downregulate moderately the Tcf/Lef-responsive promoter (Figure 6). This suggests that downregulation of Tcf activity by p53 is independent of the autoregulatory loop involving -catenin.

Figure 6.

Downregulation of Tcf-4 transcription by p53 is independent of the -catenin/p53 autoregulatory loop. C33A cells were transfected with different combinations of the plasmids for pTOPFLASH-luciferase Tcf/Lef-responsive reporter, pCMV-p53wt, pCMV-p53mut and wild-type or S33Y -catenin degradation-deficient mutant. The TCF-site-negative pFOPFLASH reporter was employed as a control. Ratios of relative light units from the pTOPFLASH- or pFOPFLASH-firefly luciferase and Renilla luciferase control from triplicate experiments with standard deviations are given

Full figure and legend (23K)

Furthermore, this experiment indicates that some of the total Tcf/Lef transcriptional activity, as represented by pTOPFLASH reporter, is repressed by p53. Even though there is a downregulation by p53 seen also with the pFOPFLASH control, this regulation is smaller than the effect on pTOPFLASH, leaving some p53-mediated downregulation dependent on the Tcf/Lef-site containing pTOPFLASH reporter (Figure 6). Taken together, it appears that p53 downregulates a significant fraction of the Tcf/Lef transcriptional activity in a cell downstream of and independent of -catenin regulation.

Discussion

p53 exerts its function in cell-cycle arrest and apoptosis induction mostly through its activity as a transcription factor (Vousden and Lu, 2002). It can act as a transcriptional activator and repressor. Generally, p53 activates transcription of proapoptotic genes and leads to the expression of genes that support cell-cycle arrest. Repression by p53 targets mostly antiapoptotic- and proliferation-supporting genes. Tcf-4 is a transcription factor that induces the expression of genes that support proliferation (Korinek et al., 1997; Roose and Clevers, 1999). We provide evidence that an increase in p53 transcriptional activity leads to a drop in Tcf-4 mRNA levels measured by two different methods (Table 1), that the Tcf-4 promoter is downregulated by wild-type p53 (Figure 1), that Tcf-4 protein is reduced in cells with high levels of p53 (Figure 2) and that downregulation of Tcf-4 expression follows with a lag phase the expression of p53 (Figure 4a). Our suggestion from these observations that Tcf-4 transcription is downregulated by p53 fits the general view that proliferation-supporting factors are repressed by the activity of p53.

A central feature of Wnt/-catenin signalling is the regulation of -catenin stability with the APC tumour suppressor protein as a key regulator. It shuttles -catenin back from the nucleus to the cytoplasm where -catenin can then be ubiquitinated and subsequently degraded by the proteasome (Henderson, 2000; Rosin-Arbesfeld et al., 2000). APC mutations are observed in more than 80% of colon cancers (Kinzler and Vogelstein, 1996). The resulting failure to shuttle -catenin out of the nucleus and to degrade it (Clevers, 2000) can lead to stable Tcf-4/-catenin complexes in the nucleus and to constitutive activation of Tcf-4 target genes (Korinek et al., 1997). However, following the observations described here, even with mutated APC and transcriptional activation by -catenin in the nucleus, p53 may still reduce this proliferation-supporting effect by downregulating Tcf-4 expression. Thereby, p53 interferes with Tcf-4 regulation downstream of the APC/-catenin control (Rosin-Arbesfeld et al., 2003). One conclusion from this would be that p53 loss of function mutations would be required in addition to the APC mutations for tumour development. Consistent with this view, p53 mutations are prevalent in colorectal carcinomas as a late event. Mutation of APC constitutes an early event, whereas p53 mutation is a late developing alteration in the adenoma/carcinoma sequence in colorectal tumorigenesis (Fearon and Vogelstein, 1990; Baker et al., 1990b; Kinzler and Vogelstein, 1996). Furthermore, the expression of wild-type p53 in colon tumour cells can suppress cell division in cells that have already acquired an altered -catenin/APC regulation (Baker et al., 1990a).

Tcf-4 downregulation by p53 may not be restricted to colon cells, since Tcf-4 and p53 are expressed in other tissues and p53 mutations occur in many tumours of different origin. Mutations of -catenin were detected in a number of tumours, including endometrial, hepatocellular, kidney, melanoma, pancreatic, thyroid, ovarian and prostate cancers (Polakis, 2000). Tcf-4 protein is found in colon, small intestine, mammary tissue and tumour cells derived from these tissues (Barker et al., 1999). Tcf-4 mRNA is expressed in all human cancer tissues tested, which included the ovary, prostate, colon, lung, stomach, oesophagus and head/neck (Barker et al., 2000). Furthermore, we found downregulation of Tcf-4 expression by p53 in all cell types tested (Figure 1b).

In addition to the regulation of Tcf-4 expression by p53 shown in this report, other connections between Wnt/-catenin signalling and p53 regulation have been documented. The influence of p53 on the Wnt signalling pathway has been observed by showing that the gene encoding Dickkopf-1, which is able to inhibit Wnt activity, can be transcriptionally upregulated by p53 (Wang et al., 2000). Another cooperation of p53 and -catenin is their interaction in an autoregulatory loop. Elevated levels of -catenin reduce p53 degradation and thereby increase p53 activity (Damalas et al., 1999). This effect is antagonized by active p53 leading to a downregulation of -catenin protein levels (Matsuzawa and Reed, 2001; Sadot et al., 2001). Now, we could demonstrate, by overexpressing p53 in the presence of a stable and therefore constitutively active -catenin mutant, that repression of Tcf-4 transcription by p53 is independent of the p53/-catenin autoregulatory loop (Figure 6). In addition, we show in this experiment that either most of the Tcf/Lef-reporter activity after activation by degradation-resistant -catenin stems from Tcf-4 transcription and is therefore repressed by p53 or that the reporter is largely activated by other Tcf/Lef proteins and these factors are also downregulated by p53.

One aspect remaining largely unresolved is the detailed mechanism by which p53 downregulates expression of the Tcf-4 promoter, in particular, since p53-binding sites could not be identified in the Tcf-4 promoter. Only very recently, it has been shown that for numerous other p53 targets the cyclin-dependent kinase inhibitor p21 is necessary for mediating p53-dependent transcriptional repression (Löhr et al., 2003). Also for promoters of other p53 target genes, alternative bindings sites and mechanisms for transcriptional regulation have been described. For instance, partial inversion of the bipartite p53-consensus-binding site is found in the MDR1 upstream region and still allows for p53 binding and leads, in contrast to a regularly oriented site, to repression of this promoter (Johnson et al., 2001). In the hepatitis B virus, genome-specific p53 binding to an enhancer leads to a disturbance of the interaction among strong activators at other DNA-binding sites, resulting in an overall drop of transcription in the presence of p53 (Ori et al., 1998). Another example is the reduction of transcriptional activity of the Bcl-2 gene via binding of p53 to the Bcl-2 promoter and through interference with activation by the Brn-3a transcription by protein–protein interaction (Budhram-Mahadeo et al., 1999). The use of an overlapping DNA-binding element by p53 with the activating factor HNF-3 leads to a repression of the alpha-fetoprotein gene promoter (Lee et al., 1999). For the cdc2 (Yun et al., 1999) and cdc25C promoters (Krause et al., 2001; Manni et al., 2001), we and others have recently shown that p53-dependent repression can be mediated through CCAAT boxes that bind the transcriptional activator NF-Y. However, scanning the nucleotide sequence of the Tcf-4 promoter reveals that it neither contains a CCAAT box nor an element conforming to the classical p53-binding consensus (el-Deiry et al., 1992), the recently identified microsatellite sequence (Contente et al., 2002) nor an inverted p53-binding site (Johnson et al., 2001). Thus it is likely that none of the above-mentioned mechanisms applies to Tcf-4 regulation. Also, ChIP experiments indicate that p53 does not bind to the Tcf-4 promoter (Figure 5). Taken together, p53 does not seem to contact Tcf-4 promoter DNA directly to downregulate the expression of this gene. The Tcf-4 promoter may also be downregulated by p53 through the upregulation of the cdk inhibitor p21 as recently published for other p53 target genes (Löhr et al., 2003). Whether Tcf-4 downregulation by p53 also requires p21 will have to be elucidated in further experiments.

In summary, with the repression of Tcf-4 transcription, we describe a new function of the p53 tumour suppressor. The downregulation of Tcf-4 transcription by p53 may help to prevent tumour formation even when upstream components of the Wnt/-catenin signalling pathway have already been mutated with the potential of constitutively activating Tcf-4-dependent transcription. By this activity, p53 may control the oncogenic activity of Tcf-4 in all tissues in which both p53 and Tcf-4 are expressed.

Materials and methods

Plasmids and cloning of the Tcf-4 promoter

The promoter of the human Tcf-4 gene was amplified and sequenced by primer walking with a GenomeWalker library (Clontech) using information from the 5'-end of the published hTcf-4 (GenBank accession number Y11306) cDNA (Korinek et al. , 1997). PCR amplification in the final step from human genomic DNA with primers 5'-GGG GTA CCG GAT ACT TTT AAT GTT TCT GAC A-3' and 5'-CTG CGC CAT GGT TTT TTC ACC CAC CAG CAG C-3', digestion with NcoI and KpnI restriction enzymes and cloning into the pGL3-Basic firefly luciferase vector (Promega) yielded the Tcf-4-Luci construct. The sequence of the human Tcf-4 promoter has been deposited in the database (GenBank accession number AF522996).

The wild-type -catenin expression plasmid was made available by Hans Clevers (Korinek et al., 1997). The S33Y-mutant -catenin expression plasmid, the pTOPFLASH-luciferase reporter plasmid and the human p53 expression plasmids, pCMV-p53wt and pCMV-p53mut, were provided by Bert Vogelstein (Baker et al., 1990a; el-Deiry et al., 1995; Korinek et al., 1997; Morin et al., 1997). The control plasmid pRL-null (Promega) contains a cDNA encoding Renilla luciferase. All the construct DNAs were purified through anion exchange columns (Qiagen) and confirmed by restriction analysis and sequencing.

Cell culture, transfection and luciferase assay

Parental HCT116 cells and HCT116 cells with targeted deletion in both p53 alleles (Bunz et al., 1998) as well as inducible cell lines D.P53 A2 (wild-type DLD-1-tet-off-p53) and 175 A4 (DLD-1-tet-off-p53 with DNA-binding mutant p53) were kindly provided by Bert Vogelstein. The inducible cell lines are derivatives of the colorectal carcinoma cell line DLD-1 that has endogenous mutant p53 alleles (Yu et al., 1999). The expression of wild-type and mutant p53 in these cells is regulated by a modified tetracycline (tet)-regulated gene expression system (tet-off system) (Gossen and Bujard, 1992; Yu et al., 1999).

NIH3T3, HFF, C33A, SaOS-2 and DLD-1 cells were cultured as described and transfected at 4 10⁴, 4 10⁴, 6 10⁴, 5 10⁴ and 10 10⁴ cells per well plated in 24-well plates, respectively (Krause et al., 2000; Haugwitz et al., 2002; Wasner et al., 2003a). Transfections using Fugene 6 (Roche) were carried out in triplicate with 400 ng Tcf-4-Luci and 25 ng of the other plasmids per assay. -catenin activation experiments employing the pTOPFLASH reporter used 5 10⁴ C33A cells in 0.5 ml per well. Cells were cultured overnight before transfection. Transfection was carried out in triplicate using 1 l Fugene 6 with 200 ng pTOPFLASH or pFOPFLASH, 200 ng wild-type or S33Y mutant -catenin construct, 100 ng p53 wild-type or mutant construct and 4 ng pRL-null vector. DNA amounts were held constant using pUC19 plasmid (Krause et al., 2000; Thiele et al., 2001).

Firefly and Renilla luciferase activities were assayed with the Dual Luciferase Assay System (Promega) and firefly luciferase was normalized to Renilla luciferase activity in order to compensate for variability in transfection efficiencies (Krause et al., 2000).

X-ray irradiation

HCT116 cells (5 10⁶) were seeded per 75 cm² tissue culture flasks 24 h before irradiation. At the Medizinisch-Experimentelles Zentrum Leipzig, cells were irradiated by a dose of 5 Gy (1.19 Gy/min) using a Gulmay D3-225 COMET MXR 226 (Gulmay Medical, England) X-ray generator filtered with treatment filter 9 (2.0 Cu and 3.0 Al). After irradiation, cells were cultured for 9, 24 or 48 h as described. Nuclear proteins were harvested afterwards (Andrews and Faller, 1991) and analysed by Western blotting.

Oligonucleotide microarray analysis

Synthesis of double-stranded cDNA, generation of biotinylated cRNA using the Enzo High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Santa Clara), hybridization to HuGeneFL arrays (Affymetrix), washing and staining as well as scanning were carried out as recommended in the Affymetrix Gene Expression Analysis Technical Manual. Signal intensities and expression changes were determined using the GeneChip Microarray Suite version 4.0 software (Affymetrix). A scaling across all probe sets of a given array was included to compensate for variations in the amount and quality of the cRNA samples and other experimental variables.

Western blotting, RNA extraction and real-time RT–PCR analysis

Western blot analyses were carried out with 60 g nuclear protein per sample from wild-type DLD-1-tet-off-p53 cells without and after the induction of the transgene and 200 g nuclear protein per sample from HCT116 cells, respectively. Proteins were separated on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane (Hybond-P; Amersham Pharmacia Biotech). Tcf-4 is known to be alternatively spliced (Duval et al., 2000). A Tcf-4 antibody was employed that detects the N-terminal part of the protein in which splice forms do not vary. Therefore, Western analyses should detect all Tcf-4 protein forms. In our experiments only the longest form of Tcf-4 was detected. For antigen detection anti-Tcf-4, a 1 : 1000 dilution of a mouse-monoclonal antibody (6H5-3; Upstate Biotechnology), p53 (1 : 2000, Do-1; Oncogene) and -actin (1 : 5000, AC-15; Sigma) antibodies together with SuperSignal West Chemiluminescent substrate (Pierce) were employed according to the manufacturer's suggestions. Signals were recorded with a Luminescent Image Analyzer LAS-100 (Fuji).

Extraction of total RNA from DLD-1-tet-off-p53 and real-time RT–PCR mRNA quantification with the LightCycler system (Roche) including calculations and primers for p21^WAF1/CIP1 and GAPDH detection have been described (Krause et al., 2000). Tcf-4-specific primers 5'-ACG AGG GCG AAC AGG AGG AG-3' and 5'-TGG GCG AGA GCG ATC CGT TG-3' (GenBank accession number Y11306) detecting all splice forms of Tcf-4 (Duval et al., 2000) were used at 1 M with 6 mM MgCl₂ on 50 ng RNA template in the LightCycler Reaction Mix SYBR Green I and RT–PCR Enzyme Mix (Roche). Each cycle of PCR included immediate denaturation at 95°C, 10 s of primer annealing (55°C) and 15 s of extension/synthesis (72°C). Product quantification was optimal at 86°C. Expression of uPAR mRNA (GenBank accession number AF257789) was analysed the same way with primers described earlier (Thiele et al., 2001), with the annealing temperature at 60°C and quantification step at 88°C specific for the LightCycler instrument. Tcf-4, uPAR and p21^WAF1/CIP1 expression was normalized to GAPDH mRNA levels (Krause et al., 2000). All cDNA products used in LightCycler mRNA quantifications were sequenced to verify their identity.

ChIP assays

ChIPs of TCF-4 and p21^WAF1/CIP1 promoter fragments were carried out essentially as described (Boyd et al., 1998; Wasner et al., 2003b). Two samples of DLD-1 cells with a tet-off-inducible wild-type p53 gene (Yu et al., 1999) were exposed to 1% formaldehyde without or after 9 h of induced p53 expression (Krause et al., 2000). Protein crosslinks were precipitated using 1 g of mouse monoclonal anti-p53 antibody (Ab-6, Calbiochem, Bad Soden, Germany). Samples were analysed with Taq DNA Polymerase (Qiagen) according to the manufacturer's suggestions with the following primers: human TCF-4 5'-CTT CAA CTC ACT CAA ATC CGA G-3' and 5'-AAA AGA CGA GTC CAA GGT GAA C-3' (GenBank accession number AF522996); and human p21^WAF1/CIP1 5'-GCA GCC AGG AGC CTG GGC CCC GG-3' and 5'-GGA CAC GCA GGG ACA CAC GCG GGC-3' (GenBank accession number U24170). PCR products were run on a 1.5% agarose gel and stained with SYBR Green (Biozym, Hess. Oldendorf, Germany).

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Acknowledgements

We thank Jana Lorenz for technical support, Jutta Jahns for assistance with the cell irradiation experiments, Matthias Dobbelstein for comments on the data and Hans Clevers and Bert Vogelstein for generously providing reagents and cells. KR and KT were recipients of graduate fellowships awarded by the Freistaat Sachsen. KE is supported by grants from the Bundesministerium für Bildung und Forschung, the Interdisciplinary Centre for Clinical Research (IZKF) at the University of Leipzig and the Deutsche Forschungsgemeinschaft.