β-Catenin and its close homologue plakoglobin (γ-catenin) are major constituents of submembranal cell-cell adhesion sites. In addition, β-catenin is a key component in the canonical Wnt pathway. Aberrantly activated β-catenin signaling contributes to cancer progression by inducing [in complex with lymphocyte enhancer factor (LEF)/T-cell factor (TCF)] the transcription of proliferation-related genes such as cyclin D1 and c-myc. Plakoglobin can also activate LEF/TCF-mediated transcription. Excessive β-catenin signaling in MEF triggers a p53-mediated antiproliferative response by inducing the expression of ARF. We have demonstrated previously that plakoglobin also exerts a tumor-suppressive effect in certain cancer cell lines. To identify genes induced by β-catenin and plakoglobin, DNA microarray analysis was carried out, and PML was among those genes of which the expression was significantly elevated by both plakoglobin and β-catenin. Activation of the PML promoter by β-catenin and plakoglobin was LEF/TCF-independent. We found that PML forms a complex with β-catenin in cells, and the two proteins colocalize in the nucleus. In addition, PML, p300, and β-catenin cooperated in transactivation of a subset of β-catenin-responsive genes including ARF and Siamois but not cyclin D1. Retroviral expression of β-catenin, plakoglobin, or PML suppressed the tumorigenicity of p53-negative human renal carcinoma cells, thus pointing to a novel antioncogenic response triggered by catenins that is mediated by the induction of PML.

β-Catenin and its homologue plakoglobin play an important role in cadherin-based adherens junctions by linking cadherin receptors to the actin cytoskeleton (1). β-Catenin also constitutes a key component of the canonical Wnt signaling pathway that determines cell fate specification during development (2). When Wnt signaling is inactive, β-catenin is rapidly degraded by the proteasome (3). Activation of Wnt signaling inhibits β-catenin degradation, resulting in its accumulation and nuclear translocation. Nuclear β-catenin mainly functions in complex with LEF4 /TCF to induce LEF/TCF target genes (4). This activity of β-catenin can be mediated by its interaction with TATA binding protein, an RNA polymerase II-associated factor (5), CBP/p300 proteins (6, 7, 8), and proteins involved in chromatin remodeling (9). The induction of target genes by β-catenin is a key step in Wnt-mediated cell specification during development (10) and also regulates cell proliferation (3).

Aberrant activation of β-catenin signaling is common in cancers of different origin and results from mutations that compromise the degradation of β-catenin (3). These mutations lead to the accumulation of β-catenin in the nucleus and activation of β-catenin-mediated transcription of target genes, including cyclin D1(11, 12) and c-myc, (13) that regulate cell proliferation and survival. Plakoglobin can also activate LEF/TCF-dependent transcription (14) and induces the c-myc gene (15), and a stabilizing mutation in plakoglobin was detected in gastric cancer (16).

Whereas β-catenin-driven transcription may result in enhanced proliferation, in MEFs, aberrant activation of β-catenin induces the accumulation of p53 (17) that provides a protective response to the oncogenic action of β-catenin. This elevation in p53 is achieved, at least in part, by induction of the ARF gene promoter (18). The increase in ARF most probably interferes with Mdm2-dependent degradation of p53, leading to the accumulation of p53 (19). Interestingly, activation of the p53 response by β-catenin can enhance the degradation of β-catenin by a negative feedback loop (20).

In this study, we report on the induction of PML gene expression in response to overexpression of β-catenin or plakoglobin. PML is a nuclear protein localized in characteristic dots, known as nuclear bodies, or PML domains that contain a number of proteins involved in the regulation of transcription, apoptosis, and cell cycle progression (21, 22). The assembly of nuclear bodies is regulated by covalent modification of their components with the ubiquitin-like protein SUMO (21). Chromosomal translocations that result in the fusion of PML with the RAR disrupt the normal organization of nuclear bodies and are characteristic of PML. The elevation in PML was implicated recently in the premature senescence induced by oncogenic Ras (23, 24). In addition, PML was shown to enhance p53-mediated transcription (25), and p53-mediated apoptosis is attenuated in PML−/− cells (26), suggesting that there is a cross-talk between PML and p53. We show here that the induction of PML expression by β-catenin or plakoglobin suppresses the tumorigenicity of renal carcinoma cells. Surprisingly, this suppression is p53-independent, thus identifying a novel pathway for catenin-mediated growth inhibition.

Construction of Plasmids.

A luciferase reporter plasmid driven by the PML promoter was constructed by cloning the PML promoter region (−746 to +150) into the pA3 reporter plasmid giving rise to PML-S. The PML 10/12 reporter plasmid contains PML promoter sequences from −78 to +121. The pS and pS01234 reporter plasmids of the Siamois promoter were kindly provided by Dr. D. Kimelman (University of Washington, Seattle, WA; Ref. 10). p20 TATA and p20 LEF were from Dr. E. Sadot (Weizmann Institute of Science, Rehovot, Israel). TOPFLASH was kindly provided by Dr. H. Clevers (University Medical Center, Utrecht, The Netherlands). The cyclin D1 promoter p-163CD1-luc construct was described previously (11). To prepare GFP-PML, the PML coding sequence was inserted into the pEGFP-C1 cloning vector (Clontech). The GFP-PML retrovirus pBabe-GFP-PML was produced by inserting GFP-PML into pBabe-puro. A retrovirus expressing human plakoglobin, pBabe-HA-plakoglobin, was prepared by inserting the human plakoglobin sequence into pBabe-puro. pBabe-β-catenin-S33Y was described previously (17). pLEF-1-VP16 and pLEF-1-DN were provided by Dr. R. Kemler (Max-Planck Institute of Immunobiology, Freiburg, Germany). The p300-HA expression vector was from H. Chen (The Salk Institute, La Jolla, CA), and the p14ARF promoter reporter was from Dr. K. Vousden (National Cancer Institute, Frederick, MD).

Cell Lines, Transfections, and Retroviral Infections.

293T, HCT116, and KCTCL60 cells were maintained in DMEM plus 10% bovine calf serum. For transactivation assays, 0.5 μg β-galactosidase plasmid was cotransfected with 0.5 μg reporter plasmids, 0.5–4 μg of catenin constructs, and 1.5 μg each of p300-HA and PML plasmids, and luciferase and β-galactosidase activities were determined (11). The retroviral infections using the pBabe-puro-based plasmids were carried out as described (17, 18). Cell cultures were selected for 7 days with 10 μg/ml puromycin.

DNA Microarray Analysis and Northern Blot Hybridization.

The UniGem-1 DNA microarray (Incyte Genomics) consisting of 9790 human expressed sequence tag clones was used to screen for genes induced by plakoglobin. The cDNA probes were synthesized from a mixture of KTCTL60 cell clones stably expressing plakoglobin, using reverse transcriptase (Superscript; Life Technologies, Inc.) and an 18-mer oligodeoxythymidylic acid primer in the presence of Cy3-dCTP, whereas the control (neor) clones were labeled with Cy5-dCTP. Image and quantitative analysis were carried out with the GEMTools software (Incyte Genomics). For Northern blot hybridization, polyadenylated RNA isolated from 250 μg total RNA using the PolyATtract system IV (Promega) was hybridized with 32P-labeled cDNA probes for PML and Sp100 obtained from Dr. A. Dejean (Institut Pasteur, Paris, France). The primer sequences used for RT-PCR of PML and cyclophilin A are available on request.

Immunofluorescence, Western Blotting, and Immunoprecipitation.

Cells were cultured on glass coverslips, fixed with 3% paraformaldehyde in PBS, and permeabilized with 0.5% Triton X-100. The coverslips were incubated with monoclonal anti-PML antibodies obtained from Dr. R. van Driel (University of Amsterdam, Amsterdam, The Netherlands), or polyclonal anti-PML antibodies from Dr. G. Maul (The Wistar Institute, Philadelphia, PA) and Dr. H. Will and K. Jensen (Universitat Hamburg, Hamburg, Germany). Anti-HA and anti-p300 polyclonal antibodies were from Santa-Cruz (Santa Cruz, CA), and monoclonal anti-HA clone 12CA5 was from Roche (Mannheim, Germany). The epifluorescence images were visualized with an Axiovert S100 TV microscope equipped with a cooled, scientific grade, charge-coupled device camera (Photometrics, Tucson, AZ) and acquired with the DeltaVision 2.10 software on a Silicon Graphics computer. The antibodies used for Western blotting were monoclonal anti-GFP (Roche), polyclonal anti-HA (Santa Cruz), monoclonal anti-β-catenin (Transduction Laboratories, Lexington, KY), and the anti p53 antibodies described by Sadot et al.(20). Immunoprecipitation was performed as described (27).

Tumorigenicity Assays.

KTCTL60 cells (106 cells/animal) expressing retrovirally transduced proteins, or the puror gene alone, were injected s.c. into 6-week-old CD1 nude male mice. Groups of five mice were followed for 3 weeks when the size of tumors in mice injected with the puror KTCTL60 cells reached ∼2 cm in diameter.

β-Catenin and Plakoglobin Induce the Expression of PML.

In a previous study we have shown that the expression of plakoglobin in a highly tumorigenic human renal carcinoma cell line, KTCTCL60, that did not express plakoglobin (14, 28), suppresses the tumorigenicity of these cells (28). To identify potential target genes responsible for this tumor suppression, we performed DNA microarray analysis comparing the expression of genes in a mixture of KTCTL60 cell clones stably expressing plakoglobin to that of a mixture of clones expressing the neor gene alone. PML and Sp100, two major components of PML nuclear bodies (21), were among the genes of which the RNA levels were significantly elevated in plakoglobin-expressing cells (Fig. 1,A). These results were confirmed by Northern blot hybridization of polyadenylated RNA with cDNA probes to PML and Sp100 (Fig. 1,B). To examine whether β-catenin can also induce the expression of PML, each of the catenins was transiently transfected into KTCTL60 cells and the level of PML determined by RT-PCR. Both β-catenin and plakoglobin induced an increase in the level of PML RNA (Fig. 1 C). Because only a fraction of the cells (∼30%) was transfected, the real increase in PML in the transfected cells was probably higher.

Activation of the PML Promoter by β-Catenin and Plakoglobin.

To address the mechanism responsible for the elevation in PML induced by catenins, we examined the response of the PML promoter to increased β-catenin or plakoglobin expression (Fig. 2). The luciferase reporter gene driven by the PML promoter (containing nucleotides −746 to +150) was activated 6-fold when β-catenin was cotransfected into 293T cells (Fig. 2,A, β-CAT Y33, black bar). A slightly lower activation of the PML promoter was also obtained by plakoglobin transfection (Fig. 2,A, PG). We identified two putative LEF/TCF sites at positions −283 (TCTTTGTACGG) and −221 (TGGATCAAAGC) of the PML promoter (Fig. 2,A), and constructed a PML reporter plasmid lacking these sites. Unexpectedly, this deletion mutant of the PML promoter was equally well activated by β-catenin or plakoglobin (Fig. 2,A, hatched bars). This suggests that activation of the PML promoter by both catenins is LEF/TCF-independent. To additionally test this possibility, we compared the capacity of a dominant-positive LEF-1-VP16 fusion protein to activate the promoters of PML and Siamois, a classical LEF/TCF target gene in Xenopus(10). Whereas the Siamois promoter was activated 12-fold by LEF-1-VP16 (Fig. 2,B, black bars), the PML promoter was not significantly affected (Fig. 2,B, white bars). β-Catenin-driven activation of both the PML promoter and the synthetic LEF/TCF reporter TOPFLASH was inhibited by the E-cadherin cytoplasmic tail (Fig. 2,C) that prevents the interaction of β-catenin with its protein partners by binding to the central arm domain of β-catenin (29). DN-LEF-1 caused a reduction in β-catenin activation of TOPFLASH (Fig. 2,C, left panel) but, in contrast, slightly enhanced the activation of the PML promoter (Fig. 2 C, right panel), pointing to different mechanisms by which β-catenin activates these two promoters. Taken together, these results suggest that activation of the PML promoter does not require the LEF/TCF sites but probably involves other proteins that bind to the arm domain of β-catenin.

PML 10/12 (Fig. 2 A) was the shortest reporter construct (−78 to +121) that retained promoter activity above the pA3 plasmid background level (data not shown). Because PML 10/12 lacks the IFN-responsive elements of the PML promoter and we did not detect binding sites for other transcription factors in PML 10/12 (the PML promoter is TATA-less), this suggests that β-catenin may operate by interacting with the basic transcriptional machinery at the PML promoter.

Cooperation between PML and β-Catenin in Transcriptional Activation.

PML and β-catenin were both shown to interact with CBP/p300, and the interaction between β-catenin and p300 increases β-catenin-mediated transactivation. Because PML colocalizes with CBP/p300 in the nucleus (30, 31) and transfected β-catenin forms nuclear aggregates (14), we examined the localization of β-catenin and PML in cells transiently transfected with β-catenin. In such cells (Fig. 3, A–C), the endogenous PML of which the level increased (Fig. 3,B) was partially recruited to these β-catenin aggregates (Fig. 3, B and C). The interaction between PML and β-catenin was also demonstrated by coimmunoprecipitation from lysates of cells cotransfected with GFP-PML and β-catenin (Fig. 3,D). To additionally investigate the localization of PML, β-catenin, and their common partner p300, small amounts of GFP-PML and β-catenin were cotransfected into 293T cells, the cells were immunostained for HA-tagged β-catenin and endogenous p300, and triple fluorescence images of GFP-PML, β-catenin, and p300 were obtained (Fig. 3, E–H). Such images indicated colocalization of the three proteins in some of the nuclear bodies (Fig. 3, E–H, arrowhead), whereas in other cases β-catenin-positive speckles and PML bodies were adjacent to each other (Fig. 3 H, arrow).

To examine whether an interaction among PML, β-catenin, and p300 affects β-catenin-mediated transcription, the activity of β-catenin-responsive gene promoters was analyzed in the presence or absence of cotransfected PML and p300 (Fig. 4). Activation (by β-catenin) of an artificial LEF/TCF reporter plasmid (Fig. 4,A, p20 LEF) was only slightly increased by PML or p300 but was significantly augmented by a combination of the two proteins, in contrast to a control reporter plasmid of which the transcription remained low in the presence of the three proteins (Fig. 4,A, p20 TATA). A similar result was obtained using the Siamois gene promoter (Fig. 4,B). In this experiment, the amount of transfected β-catenin was reduced to achieve an ∼2-fold activation of the Siamois promoter (Fig. 4,B). This activation was only mildly increased in the presence of p300 or PML when each protein was added alone to β-catenin (Fig. 4,B). The response of the Siamois promoter to β-catenin was strongly enhanced when PML was added together with p300, similarly to the response of p20 LEF. In the absence of β-catenin, neither PML nor p300 could affect the basal activity of the Siamois promoter, suggesting that PML and p300 cooperate with β-catenin in a specific manner. A mutant Siamois promoter, lacking LEF/TCF sites (Ref. 10; Fig. 4,B, marked S), was not activated by β-catenin, and only weakly activated by β-catenin plus p300 (presumably via a LEF/TCF-independent mechanism). This activation did not respond to PML (Fig. 4 B, white bars), demonstrating a specific role for PML in transactivation mediated by the β-catenin-LEF/TCF complex.

Next, we examined whether the cooperation between β-catenin and PML plays a role in β-catenin-dependent growth suppression by enhancing the activation of genes conferring growth arrest. We used the ARF gene promoter as a target of β-catenin displaying growth inhibitory functions, because transcription of ARF is induced by β-catenin in MEF and confers growth arrest (18). The ability of PML to affect this induction of the ARF promoter was compared it to its effect on the cyclin D1 promoter. Whereas activation of the ARF promoter by β-catenin was significantly enhanced by PML in the presence of p300 (Fig. 4,C), the response of the cyclin D1 promoter to β-catenin was enhanced by p300 but was not additionally elevated by PML (Fig. 4 D).

β-Catenin, Plakoglobin, and PML Suppress the Tumorigenicity of Renal Carcinoma Cells.

Because KTCTL60 renal carcinoma cells responded to β-catenin and plakoglobin overexpression by inducing PML (Fig. 1), we used these cells to address the biological significance of PML elevation induced by β-catenin and plakoglobin. KTCTL60 cell pools stably expressing β-catenin, plakoglobin, or GFP-PML were produced by retroviral infection, and expression of the infected proteins was confirmed by Western blotting (Fig. 5,A). Because the antibodies against PML did not detect the endogenous PML on Western blots, the increase in PML in cells expressing catenins was verified by RT-PCR (results not shown). These cells were injected into nude mice to follow tumor development. Twenty days after injection, the tumors formed by cells expressing β-catenin, plakoglobin, or PML (Fig. 5, B and C) were significantly smaller than those formed by cells expressing the puror gene alone (Fig. 5 C).

β-Catenin was shown previously to elicit a p53-mediated growth arrest in MEF (18). Therefore, we determined whether the tumor suppressive effect described above is modulated by p53. KTCTL60 cells, unlike HCT116 human colon cancer cells, did not display detectable levels of p53 either before or after the induction of DNA damage by doxorubicin or cisplatin (Fig. 5, D and E), nor in the presence of the proteasome inhibitor MG132 that stabilizes p53 (Fig. 5 E). Hence, the tumor-suppressive effect of catenins and PML in these renal carcinoma cells is mediated by a p53-independent mechanism.

Excessive activation of oncogenes, including c-myc, ras, E2F1, and E1A, was shown to elicit a protective, antiproliferative response involving the induction of the growth-suppressor p53 gene (19). Expression of a mutant, oncogenic β-catenin in MEF also induces p53, and causes growth arrest and a senescence-like phenotype (17, 18). In this study, we have shown that the PML gene is transcriptionally activated by both β-catenin and plakoglobin in a renal carcinoma cell line, and β-catenin, plakoglobin, and PML can suppress the tumorigenicity of these cells. Whereas overexpression of PML can cause tumor suppression (32, 33) and elevated PML triggered by oncogenic Ras induces a senescence-like growth arrest (23, 24), in this study we show for the first time that transcriptional activation of PML by an oncogenic signal (elicited by catenins) can lead to tumor suppression in the absence of p53.

Activation of the PML Promoter by β-Catenin and Plakoglobin.

We demonstrated that the promoter of PML is efficiently activated by β-catenin and plakoglobin, but surprisingly, the putative LEF/TCF sites in the PML promoter were not required for this activation, and DN-LEF-1 failed to inhibit β-catenin-mediated activation of the PML promoter. Most known β-catenin target genes require LEF/TCF factors for their activation (4), but β-catenin can also activate certain genes independently of LEF/TCF. For example, the promoter of WISP-1 is activated by β-catenin via CRE sites (34), and β-catenin can form a complex with the RARα to activate RAR target genes (35). Because we did not detect binding sites for cAMP response element binding protein or hormone receptors in the minimal PML promoter, the mechanism of PML promoter activation by β-catenin is most probably different from those described previously. It may possibly involve interactions between β-catenin and components of the RNA polymerase II complex (5), providing an example for promoter-specific activation via the general transcriptional machinery. This view is supported by studies showing that expression of a DN Brg-1, a general component of chromatin remodeling complexes, selectively inhibits β-catenin target gene transcription (9). Thus, β-catenin might regulate promoter-specific gene activation that is mediated by the RNA polymerase II complex.

Cooperation among β-Catenin, p300, and PML.

The involvement of p300/CBP in β-catenin-dependent transactivation (6, 7, 8, 36) and the functional interaction between PML and CBP (30) prompted us to investigate a possible cooperation among β-catenin, p300, and PML in transactivation of β-catenin target genes. We have shown that PML enhances the stimulatory effect of p300 on β-catenin-driven transcription. Previous studies demonstrated that PML activates the transcription of some genes, whereas repressing the activity of others. The positive role of PML in β-catenin-driven transactivation resembles the activation of MHC genes by PML (37) and its coactivating role in nuclear receptor-driven transcription (30, 38), in contrast to PML-mediated repression of Mad(39) and Sp1(40) target genes. In agreement with previous reports, we detected a colocalization of PML, p300 (31), and β-catenin (36) in PML bodies. We extended these observations and showed (by coimmunoprecipitation) the formation of a complex between β-catenin and PML in cells. Whereas this complex between PML and β-catenin (likely including p300/CBP) is probably responsible for the cooperation between these proteins in transcription, the requirement of PML bodies for these effects of PML is presently unknown. The exact role(s) of PML bodies in transcription is still under investigation (21, 22), and it is unclear yet whether a subset of PML bodies or the nucleoplasmic pool of PML is the one responsible for cooperation with β-catenin in transactivation. In this respect, it is noteworthy that SUMOylatim-related modification of LEF-1 inhibits β-catenin/LEF-1-mediated transactivation and leads to the sequestration of LEF-1 to nuclear bodies (41). Because, in contrast, we found that PML coactivates the β-catenin/LEF-1 complex, it is likely that this activation occurs outside the nuclear bodies. The cooperation between PML and β-catenin was very efficient in enhancing the transcription of the Siamois and ARF promoters but did not increase the activation of the cyclin D1 promoter by β-catenin. The molecular mechanism(s) underlying the promoter-specific cooperation between β-catenin and PML require additional study. It will be interesting to examine whether subsets of β-catenin target genes alter their responsiveness to β-catenin during tumor progression and whether the induction of PML expression by catenins contributes to such changes.

The Role of β-Catenin, Plakoglobin, and PML in Tumorigenesis.

In a previous report we demonstrated that overexpression of plakoglobin in KTCTL60 renal carcinoma cells results in tumor suppression (28). Here, we provide a possible molecular explanation for this phenomenon by showing that this involves the induction (by plakoglobin) of the tumor suppressor gene PML. The ability of β-catenin to induce PML and suppress tumorigenicity in the same cell line highlights the similarity in the function of the two catenins (42). β-Catenin and plakoglobin were both shown to also transform RK3E cells (15). The tumor suppression we observed in KTCTL60 cells after overexpression of plakoglobin or β-catenin may provide a protective cellular mechanism against cancer counteracting the oncogenic/proliferative effects of catenins (Fig. 6). Such protective mechanism (against β-catenin) was demonstrated in MEF where it is mediated by the growth-suppressive effect elicited by p53 (18). Whereas KTCTL60 cells do not express p53, β-catenin-mediated induction of PML may result in the cooperation of PML with β-catenin in transactivation of the ARF promoter, thus leading (in cells possessing functional p53) to an elevation in p53 (Fig. 6). This may constitute an additional mechanism to the known capacity of PML to promote downstream effects of the p53 pathway (Refs. 25, 26; Fig. 6). The antitumorigenic response elicited by catenin overexpression described in this study occurs in KTCTL60 cells that are highly tumorigenic and lack p53, suggesting that p53-independent protective mechanisms against oncogenic signaling by catenins also exist (Fig. 6). Whereas the elevation of β-catenin was extensively documented in various cancers (3), several studies also reported on the loss of catenins during tumor progression (43, 44). This loss was usually associated with a reduction in E-cadherin and was interpreted to result in the promotion of the metastatic process. We propose, in addition, that the loss of catenin expression in tumors that preserve a p53-independent growth-suppressive response to catenin signaling may enhance tumor progression by inactivating this defense mechanism.

Fig. 1.

Induction of PML and Sp100 by plakoglobin and β-catenin. A, three independent human renal carcinoma cell (KTCTL60) clones stably expressing plakoglobin (PG), and three expressing the neor gene alone, were used to prepare RNA that was tested, after Cy3 (PG) and Cy5 (neor) labeling (after reverse transcription), for expression of genes using an Incyte DNA microarray. Hybridization signal intensity and fold induction of PML and Sp100 expression in PG-transfected cells compared with neor controls is shown. B, Northern blot hybridization determining PML and Sp100 levels in renal carcinoma cells stably expressing plakoglobin or the neor genes. Glyceraldehyde-3-phosphate dehydrogenase served as loading control. C, transient transfection of plakoglobin (PG) or β-catenin (β-cat) into renal carcinoma cells was followed by RT-PCR to examine changes in the level of PML RNA. Cyclophilin A RNA level served as control. Expression of transfected HA-tagged plakoglobin and β-catenin in duplicate dishes was determined by Western blotting (WB) with anti-HA antibody.

Fig. 1.

Induction of PML and Sp100 by plakoglobin and β-catenin. A, three independent human renal carcinoma cell (KTCTL60) clones stably expressing plakoglobin (PG), and three expressing the neor gene alone, were used to prepare RNA that was tested, after Cy3 (PG) and Cy5 (neor) labeling (after reverse transcription), for expression of genes using an Incyte DNA microarray. Hybridization signal intensity and fold induction of PML and Sp100 expression in PG-transfected cells compared with neor controls is shown. B, Northern blot hybridization determining PML and Sp100 levels in renal carcinoma cells stably expressing plakoglobin or the neor genes. Glyceraldehyde-3-phosphate dehydrogenase served as loading control. C, transient transfection of plakoglobin (PG) or β-catenin (β-cat) into renal carcinoma cells was followed by RT-PCR to examine changes in the level of PML RNA. Cyclophilin A RNA level served as control. Expression of transfected HA-tagged plakoglobin and β-catenin in duplicate dishes was determined by Western blotting (WB) with anti-HA antibody.

Close modal
Fig. 2.

Activation of the PML promoter by β-catenin and plakoglobin does not require cooperation with LEF/TCF. A, a PML promoter reporter plasmid containing two putative LEF/TCF sites (PML-S), a shorter promoter construct lacking these sites (PML-10/12), and the empty pA3 reporter plasmid were transfected into 293T cells in the absence or presence of an active β-catenin mutant (β-CAT Y33) or plakoglobin (PG), and luciferase activity was determined in duplicate dishes and normalized for transfection efficiency by cotransfected β-galactosidase. Fold activation was calculated relative to luciferase activity in the absence of β-catenin. B, activation of the Siamois promoter, but not the PML promoter, by a dominant-positive LEF-1-VP16 chimeric protein. The reporter plasmids were transfected into 293T cells in the absence or presence of increasing amounts (0.1–1.0 μg) of LEF-1-VP16 plasmid. C, dominant-negative LEF-1 (DN-LEF-1) does not inhibit the activation of the PML promoter by β-catenin. The PML promoter plasmid (PML-S, right panel) or a synthetic LEF/TCF-responsive plasmid (TOPFLASH, left panel) were transfected with or without β-catenin and DN-LEF-1, or the cadherin cytoplasmic tail. Note that whereas the cadherin cytoplasmic tail (that sequesters away β-catenin from LEF/TCF) inhibited the activation of both TOPFLASH and the PML promoter, DN-LEF-1 only inhibited the activation of TOPLFLASH, but not the PML promoter, indicating that PML promoter activation by β-catenin is LEF/TCF-independent; bars, ±SD.

Fig. 2.

Activation of the PML promoter by β-catenin and plakoglobin does not require cooperation with LEF/TCF. A, a PML promoter reporter plasmid containing two putative LEF/TCF sites (PML-S), a shorter promoter construct lacking these sites (PML-10/12), and the empty pA3 reporter plasmid were transfected into 293T cells in the absence or presence of an active β-catenin mutant (β-CAT Y33) or plakoglobin (PG), and luciferase activity was determined in duplicate dishes and normalized for transfection efficiency by cotransfected β-galactosidase. Fold activation was calculated relative to luciferase activity in the absence of β-catenin. B, activation of the Siamois promoter, but not the PML promoter, by a dominant-positive LEF-1-VP16 chimeric protein. The reporter plasmids were transfected into 293T cells in the absence or presence of increasing amounts (0.1–1.0 μg) of LEF-1-VP16 plasmid. C, dominant-negative LEF-1 (DN-LEF-1) does not inhibit the activation of the PML promoter by β-catenin. The PML promoter plasmid (PML-S, right panel) or a synthetic LEF/TCF-responsive plasmid (TOPFLASH, left panel) were transfected with or without β-catenin and DN-LEF-1, or the cadherin cytoplasmic tail. Note that whereas the cadherin cytoplasmic tail (that sequesters away β-catenin from LEF/TCF) inhibited the activation of both TOPFLASH and the PML promoter, DN-LEF-1 only inhibited the activation of TOPLFLASH, but not the PML promoter, indicating that PML promoter activation by β-catenin is LEF/TCF-independent; bars, ±SD.

Close modal
Fig. 3.

Complex formation between β-catenin and PML and colocalization of p300, PML, and β-catenin in nuclear bodies. A–C, overexpression of β-catenin (β-CAT) into 293T cells results in the formation of nuclear aggregates consisting of β-catenin (A) that recruit part of the endogenous PML (B) into these structures (C). D, coimmunoprecipitation (IP) of GFP-PML from cells transfected with both GFP-PML and β-catenin demonstrated their participation in the same molecular complex. E–H, transfection of small amounts of both GFP-PML (F) and HA-tagged β-catenin (E) showed that part of the endogenous p300 molecules (G) were recruited to nuclear bodies containing all three molecules (H, arrowhead). In other cases, β-catenin and PML bodies were found adjacent to each other (H, arrow). PML was detected by the GFP tag, whereas β-catenin and p300 were visualized by secondary antibodies labeled with Cy3 and Cy5. The bar represents 10 μm. Note the increase in endogenous PML level in the β-catenin-transfected cell (B) compared with nontransfected neighboring cells. Note also that the grainy distribution of p300 in nontransfected cells (G) was altered in the two transfected cells (G, arrow and arrowhead) where p300 was more efficiently recruited to nuclear bodies.

Fig. 3.

Complex formation between β-catenin and PML and colocalization of p300, PML, and β-catenin in nuclear bodies. A–C, overexpression of β-catenin (β-CAT) into 293T cells results in the formation of nuclear aggregates consisting of β-catenin (A) that recruit part of the endogenous PML (B) into these structures (C). D, coimmunoprecipitation (IP) of GFP-PML from cells transfected with both GFP-PML and β-catenin demonstrated their participation in the same molecular complex. E–H, transfection of small amounts of both GFP-PML (F) and HA-tagged β-catenin (E) showed that part of the endogenous p300 molecules (G) were recruited to nuclear bodies containing all three molecules (H, arrowhead). In other cases, β-catenin and PML bodies were found adjacent to each other (H, arrow). PML was detected by the GFP tag, whereas β-catenin and p300 were visualized by secondary antibodies labeled with Cy3 and Cy5. The bar represents 10 μm. Note the increase in endogenous PML level in the β-catenin-transfected cell (B) compared with nontransfected neighboring cells. Note also that the grainy distribution of p300 in nontransfected cells (G) was altered in the two transfected cells (G, arrow and arrowhead) where p300 was more efficiently recruited to nuclear bodies.

Close modal
Fig. 4.

Cooperation among β-catenin, p300, and PML in the activation of the Siamois and ARF promoter, but not the cyclin D1 promoter. A, β-catenin (β-CAT Y33) was transfected into 293T cells together with a reporter plasmid containing three LEF binding sites (p20 LEF) or with a plasmid lacking the LEF sites (p20 TATA). Activation of these plasmids was determined in different combinations of p300, PML, and β-catenin. B, a similar experiment was carried out with plasmids containing the Siamois promoter (S01234) and a Siamois promoter with mutated LEF sites (S). Activation of ARF (C) and cyclin D1 (D) promoters was tested in different combinations of β-catenin, PML, and p300. Note that whereas activation (by β-catenin) of the synthetic p20 LEF reporter, and the Siamois and ARF promoters was cooperatively enhanced by the combination of PML and p300 (last dark column on the right), the activation of cyclin D1 was not increased by PML; bars, ±SD.

Fig. 4.

Cooperation among β-catenin, p300, and PML in the activation of the Siamois and ARF promoter, but not the cyclin D1 promoter. A, β-catenin (β-CAT Y33) was transfected into 293T cells together with a reporter plasmid containing three LEF binding sites (p20 LEF) or with a plasmid lacking the LEF sites (p20 TATA). Activation of these plasmids was determined in different combinations of p300, PML, and β-catenin. B, a similar experiment was carried out with plasmids containing the Siamois promoter (S01234) and a Siamois promoter with mutated LEF sites (S). Activation of ARF (C) and cyclin D1 (D) promoters was tested in different combinations of β-catenin, PML, and p300. Note that whereas activation (by β-catenin) of the synthetic p20 LEF reporter, and the Siamois and ARF promoters was cooperatively enhanced by the combination of PML and p300 (last dark column on the right), the activation of cyclin D1 was not increased by PML; bars, ±SD.

Close modal
Fig. 5.

Suppression of tumorigenesis in renal carcinoma cells after retroviral-mediated infection of PML, β-catenin, or plakoglobin is independent of p53. A, KTCTL60 human renal carcinoma cells were infected with pBABE retroviral vectors containing HA-tagged β-catenin, plakoglobin, or GFP-tagged PML, and selected for puromycin resistance. Pools of puror cells were analyzed by Western blotting for expression of the transgenes using anti HA and GFP antibodies. B, cells were injected into nude mice, and development of tumors after 3 weeks is presented. C, the weight of the excised tumors is presented as the mean; bars, ±SE. Note the similar dramatic suppression of tumor growth by β-catenin, plakoglobin, and PML. D, expression of p53 was determined by Western blotting with anti-p53 antibodies in KTCTL60 cells and HCT116 colon carcinoma cells after treatment for 24 or 48 h with doxorubicin (DOX), or for 24 h with cisplatin (E), or for 4 h with the proteasome inhibitor MG132. The level of tubulin served as loading control. Note that whereas HCT116 cells displayed an increase in p53 levels after DNA damage caused by doxorubicin or cisplatin, or after inhibiting proteasomal degradation by MG132, KTCTL60 showed no detectable p53 levels under these conditions, indicating the absence of p53 in these cells.

Fig. 5.

Suppression of tumorigenesis in renal carcinoma cells after retroviral-mediated infection of PML, β-catenin, or plakoglobin is independent of p53. A, KTCTL60 human renal carcinoma cells were infected with pBABE retroviral vectors containing HA-tagged β-catenin, plakoglobin, or GFP-tagged PML, and selected for puromycin resistance. Pools of puror cells were analyzed by Western blotting for expression of the transgenes using anti HA and GFP antibodies. B, cells were injected into nude mice, and development of tumors after 3 weeks is presented. C, the weight of the excised tumors is presented as the mean; bars, ±SE. Note the similar dramatic suppression of tumor growth by β-catenin, plakoglobin, and PML. D, expression of p53 was determined by Western blotting with anti-p53 antibodies in KTCTL60 cells and HCT116 colon carcinoma cells after treatment for 24 or 48 h with doxorubicin (DOX), or for 24 h with cisplatin (E), or for 4 h with the proteasome inhibitor MG132. The level of tubulin served as loading control. Note that whereas HCT116 cells displayed an increase in p53 levels after DNA damage caused by doxorubicin or cisplatin, or after inhibiting proteasomal degradation by MG132, KTCTL60 showed no detectable p53 levels under these conditions, indicating the absence of p53 in these cells.

Close modal
Fig. 6.

Molecular mechanisms for the oncogenic and tumor-suppressive effects elicited by activation of β-catenin signaling. Oncogenic mutations in components of the Wnt pathway lead to accumulation of β-catenin (β-CAT) and aberrant activation (by β-catenin together with LEF/TCF) of target genes that promote cell proliferation, including cyclin D1 and c-myc, thus leading to development of cancer. β-Catenin also induces the expression of the PML gene, which promotes growth inhibition or senescence, either independently of β-catenin, or by coactivating together with β-catenin and p300 the transcription of growth-suppressive genes such as ARF (and perhaps other genes). The antitumorigenic response elicited by β-catenin may involve the accumulation of p53 by increasing ARF in some cases or may act independently of p53.

Fig. 6.

Molecular mechanisms for the oncogenic and tumor-suppressive effects elicited by activation of β-catenin signaling. Oncogenic mutations in components of the Wnt pathway lead to accumulation of β-catenin (β-CAT) and aberrant activation (by β-catenin together with LEF/TCF) of target genes that promote cell proliferation, including cyclin D1 and c-myc, thus leading to development of cancer. β-Catenin also induces the expression of the PML gene, which promotes growth inhibition or senescence, either independently of β-catenin, or by coactivating together with β-catenin and p300 the transcription of growth-suppressive genes such as ARF (and perhaps other genes). The antitumorigenic response elicited by β-catenin may involve the accumulation of p53 by increasing ARF in some cases or may act independently of p53.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by grants from the Israel Science Foundation and the M.D. Morros Institute For Cancer Research.

4

The abbreviations used are: LEF, lymphocyte enhancer factor; ARF, alternative reading frame; CBP, cAMP-responsive element binding protein binding protein; CRE, cAMP response element; DN, dominant-negative; GFP, green fluorescent protein; HA, hemagglutinin; MEF, mouse embryo fibroblast; PML, promyelocytic leukemia; RAR, retinoic acid receptor; RT-PCR, reverse transcription-PCR; SUMO, small ubiquitin-related modifier; TCF, T-cell factor.

We thank E. Feinstein and E. Paz (QBI Enterprises LTD, Nes Ziona, Israel) for the DNA microarray analysis, and A. Dejean, G. Maul, R. van Driel, R. Kemler, and H. Will for antibodies and plasmids.

1
Ben-Ze’ev A., Geiger B. Differential molecular interactions of β-catenin and plakoglobin in adhesion, signaling and cancer.
Curr. Opin. Cell. Biol.
,
10
:
629
-639,  
1998
.
2
Wodarz A., Nusse R. Mechanisms of Wnt signaling in development.
Annu. Rev. Cell. Dev. Biol.
,
14
:
59
-88,  
1998
.
3
Polakis P. Wnt signaling and cancer.
Genes Dev.
,
14
:
1837
-1851,  
2000
.
4
Roose J., Clevers H. TCF transcription factors: molecular switches in carcinogenesis.
Biochim. Biophys. Acta
,
1424
:
M23
-M37,  
1999
.
5
Hecht A., Litterst C., Huber O., Kemler R. Functional characterization of multiple transactivating elements in β-catenin, some of which interact with the TATA-binding protein in vitro.
J. Biol. Chem.
,
274
:
18017
-18025,  
1999
.
6
Hecht A., Vleminckx K., Stemmler M., van Roy F., Kemler R. The p300/CBP acetyltransferases function as transcriptional coactivators of β-catenin in vertebrates.
EMBO J.
,
19
:
1839
-1850,  
2000
.
7
Takemaru K., Moon R. The transcriptional coactivator CBP interacts with β-catenin to activate gene expression.
J. Cell. Biol.
,
149
:
249
-254,  
2000
.
8
Sun Y., Kolligs F. T., Hottiger M. O., Mosavin R., Fearon E. R., Nabel G. J. Regulation of β-catenin transformation by the p300 transcriptional coactivator.
Proc. Natl. Acad. Sci. USA
,
97
:
12613
-12618,  
2000
.
9
Barker N., Hurlstone A., Musisi H., Miles A., Bienz M., Clevers H. The chromatin remodelling factor Brg-1 interacts with β-catenin to promote target gene activation.
EMBO J.
,
20
:
4935
-4943,  
2001
.
10
Brannon M., Gomperts M., Sumoy L., Moon R. T., Kimelman D. A β-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus.
Genes Dev.
,
11
:
2359
-2370,  
1997
.
11
Shtutman M., Zhurinsky J., Simcha I., Albanese C., D’Amico M., Pestell R., Ben-Ze’ev A. The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway.
Proc. Natl. Acad. Sci. USA
,
96
:
5522
-5527,  
1999
.
12
Tetsu O., McCormick F. β-catenin regulates expression of cyclin D1 in colon carcinoma cells.
Nature (Lond.)
,
398
:
422
-426,  
1999
.
13
He T., Sparks A., Rago C., Hermeking H., Zawel L., da Costa L., Morin P., Vogelstein B., Kinzler K. Identification of c-MYC as a target of the APC pathway.
Science (Wash. DC)
,
281
:
1509
-1512,  
1998
.
14
Simcha I., Shtutman M., Salomon D., Zhurinsky J., Sadot E., Geiger B., Ben-Ze’ev A. Differential nuclear translocation and transactivation potential of β-catenin and plakoglobin.
J. Cell Biol.
,
141
:
1433
-1448,  
1998
.
15
Kolligs F., Kolligs B., Hajra K., Hu G., Tani M., Cho K., Fearon E. γ-Catenin is regulated by the APC tumor suppressor and its oncogenic activity is distinct from that of β-catenin.
Genes Dev.
,
14
:
1319
-1331,  
2000
.
16
Caca K., Kolligs F., Ji X., Hayes M., Qian J., Yahanda A., Rimm D., Costa J., Fearon E. β- and γ-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer.
Cell Growth Differ.
,
10
:
369
-376,  
1999
.
17
Damalas A., Ben-Ze’ev A., Simcha I., Shtutman M., Leal J., Zhurinsky J., Geiger B., Oren M. Excess β-catenin promotes accumulation of transcriptionally active p53.
EMBO J.
,
18
:
3054
-3063,  
1999
.
18
Damalas A., Kahan S., Shtutman M., Ben-Ze’ev A., Oren M. Deregulated β-catenin induces a p53- and ARF-dependent growth arrest and cooperates with Ras in transformation.
EMBO J.
,
20
:
4912
-4922,  
2001
.
19
Sherr C. J., Weber J. D. The ARF/p53 pathway.
Curr. Opin. Genet. Dev.
,
10
:
94
-99,  
2000
.
20
Sadot E., Geiger B., Oren M., Ben-Ze’ev A. Down-regulation of β-catenin by activated p53.
Mol. Cell. Biol.
,
21
:
6768
-6781,  
2001
.
21
Negorev D., Maul G. Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot.
Oncogene
,
20
:
7234
-7242,  
2001
.
22
Zhong S., Salomoni P., Pandolfi P. The transcriptional role of PML and the nuclear body.
Nat. Cell Biol.
,
2
:
E85
-E90,  
2000
.
23
Ferbeyre G., de Stanchina E., Querido E., Baptiste N., Prives C., Lowe S. PML is induced by oncogenic ras and promotes premature senescence.
Genes Dev.
,
14
:
2015
-2027,  
2000
.
24
Pearson M., Carbone R., Sebastiani C., Cioce M., Fagioli M., Saito S., Higashimoto Y., Appella E., Minucci S., Pandolfi P., Pelicci P. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras.
Nature (Lond.)
,
406
:
207
-210,  
2000
.
25
Fogal V., Gostissa M., Sandy P., Zacchi P., Sternsdorf T., Jensen K., Pandolfi P., Will H., Schneider C., Del Sal G. Regulation of p53 activity in nuclear bodies by a specific PML isoform.
EMBO J.
,
19
:
6185
-6195,  
2000
.
26
Guo A., Salomoni P., Luo J., Shih A., Zhong S., Gu W., Paolo Pandolfi P. The function of PML in p53-dependent apoptosis.
Nat. Cell Biol.
,
2
:
730
-736,  
2000
.
27
Zhurinsky J., Shtutman M., Ben-Ze’ev A. Differential mechanisms of LEF/TCF family-dependent transcriptional activation by β-catenin and plakoglobin.
Mol. Cell. Biol.
,
20
:
4238
-4252,  
2000
.
28
Simcha I., Geiger B., Yehuda-Levenberg S., Salomon D., Ben-Ze’ev A. Suppression of tumorigenicity by plakoglobin: an augmenting effect of N-cadherin.
J. Cell. Biol.
,
133
:
199
-209,  
1996
.
29
Sadot E., Simcha I., Shtutman M., Ben-Ze’ev A., Geiger B. Inhibition of β-catenin-mediated transactivation by cadherin derivatives.
Proc. Natl. Acad. Sci. USA
,
95
:
15339
-15344,  
1998
.
30
Doucas V., Tini M., Egan D., Evans R. Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling.
Proc. Natl. Acad. Sci. USA
,
96
:
2627
-2632,  
1999
.
31
Boisvert F., Kruhlak M., Box A., Hendzel M., Bazett-Jones D. The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.
J. Cell Biol.
,
152
:
1099
-1106,  
2001
.
32
Koken M., Linares-Cruz G., Quignon F., Viron A., Chelbi-Alix M., Sobczak-Thepot J., Juhlin L., Degos L., Calvo F., de The H. The PML growth-suppressor has an altered expression in human oncogenesis.
Oncogene
,
10
:
1315
-1324,  
1995
.
33
Le X., Vallian S., Mu Z., Hung M., Chang K. Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis.
Oncogene
,
16
:
1839
-1849,  
1998
.
34
Xu L., Corcoran R. B., Welsh J. W., Pennica D., Levine A. J. WISP-1 is a Wnt-1- and β-catenin-responsive oncogene.
Genes Dev.
,
14
:
585
-595,  
2000
.
35
Tice D., Szeto W., Soloviev I., Rubinfeld B., Fong S., Dugger D., Winer J., Williams P., Wieand D., Smith V., Schwall R., Pennica D., Polakis P. Synergistic induction of tumor antigens by wnt-1 signaling and retinoic acid revealed by gene expression profiling.
J. Biol. Chem.
,
277
:
14329
-14335,  
2002
.
36
Miyagishi M., Fujii R., Hatta M., Yoshida E., Araya N., Nagafuchi A., Ishihara S., Nakajima T., Fukamizu A. Regulation of lef-mediated transcription and p53-dependent pathway by associating β-catenin with CBP/p300.
J. Biol. Chem.
,
275
:
35170
-35175,  
2000
.
37
Zheng P., Guo Y., Niu Q., Levy D., Dyck J., Lu S., Sheiman L., Liu Y. Proto-oncogene PML controls genes devoted to MHC class I antigen presentation.
Nature (Lond.)
,
396
:
373
-376,  
1998
.
38
Zhong S., Delva L., Rachez C., Cenciarelli C., Gandini D., Zhang H., Kalantry S., Freedman L., Pandolfi P. A RA-dependent, tumour-growth suppressive transcription complex is the target of the PML-RARα and T18 oncoproteins.
Nat. Genet.
,
:
287
-295,  
1999
.
39
Khan M., Nomura T., Kim H., Kaul S., Wadhwa R., Shinagawa T., Ichikawa-Iwata E., Zhong S., Pandolfi P., Ishii S. Role of PML and PML-RARα in Mad-mediated transcriptional repression.
Mol. Cell
,
7
:
1233
-1243,  
2001
.
40
Vallian S., Chin K., Chang K. The promyelocytic leukemia protein interacts with Sp1 and inhibits its transactivation of the epidermal growth factor receptor promoter.
Mol. Cell. Biol.
,
18
:
7147
-7156,  
1998
.
41
Sachdev S., Bruhn L., Sieber H., Pichler A., Melchior F., Grosschedl R. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies.
Genes Dev.
,
15
:
3088
-3103,  
2001
.
42
Zhurinsky J., Shtutman M., Ben-Ze’ev A. Plakoglobin and β-catenin: protein interactions, regulation and biological roles.
J. Cell Sci.
,
113
:
3127
-3139,  
2000
.
43
Amitay R., Nass D., Meitar D., Goldberg I., Davidson B., Trakhtenbrot L., Brok-Simoni F., Ben-Ze’ev A., Rechavi G., Kaufmann Y. Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma.
Am. J. Pathol.
,
159
:
43
-49,  
2001
.
44
Pirinen R., Hirvikoski P., Johansson R., Hollmen S., Kosma V. Reduced expression of α-catenin, β-catenin, and γ-catenin is associated with high cell proliferative activity and poor differentiation in non-small cell lung cancer.
J. Clin. Pathol.
,
54
:
391
-395,  
2001
.