Inactivation of the adenomatous polyposis coli(APC) gene is a critical event in the development of human colorectal cancers. At the biochemical level, several functions have been assigned to the multidomain APC protein, but the cellular effects of APC expression and how they relate to its biochemical functions are less well defined. To address these issues, we generated a recombinant adenovirus (Ad-CBR) that constitutively expresses the central third of APC, which includes all of the known β-catenin binding repeats. When expressed in colon cancer cells, Ad-CBR blocked the nuclear translocation of β-catenin and inhibitedβ-catenin/Tcf-4-mediated transactivation. Accordingly, expression of endogenous targets of the APC/β-catenin/Tcf-4 pathway was down-regulated. Ad-CBR infection of colorectal cancer cell lines with mutant APC but wild-type β-catenin resulted in substantial growth arrest followed by apoptosis. These effects were attenuated in lines with wild-type APC but with mutatedβ-catenin. These findings suggest that the β-catenin-binding domain in the central third of APC is sufficient for its tumor suppressor activity.

Inactivation of the APC3gene plays a critical and early role in the development of inherited and sporadic forms of colorectal cancer (reviewed in Ref.1). Germ-line mutations of APC result in multiple intestinal tumors in both humans and rodents. Somatic mutations of APC, occur in the majority of human colorectal cancers as well as in their benign precursor adenomas. Despite the critical role of APC in the development of colorectal tumors, the cellular and molecular mechanisms underlying its tumor suppression are not well defined. At the cellular level, a better understanding of APC function has been hindered by the inability to readily restore APC function in colorectal cancer cells (2, 3). To date, APC expression has only been successfully restored to a single human cancer cell line with mutant APC, where it was found to promote apoptosis (2).

The molecular characterization of APC function is complicated by the fact that APC contains multiple functional domains that interact with a variety of cytoplasmic proteins. These include β-catenin (4, 5), γ-catenin (6, 7), GSK3-β (8),AXIN family proteins (9, 10, 11), EB-1 (12),microtubules (13, 14), and the human homologue of the Drosophila tumor suppressor gene discs large (hDLG; Ref.15). Among these factors, the study of interaction between APC protein and β-catenin provides the most penetrating insights into APC function (4, 5). The β-catenin regulatory domain in the cAPC is removed or truncated by the majority of both inherited and somatic mutations. These truncated forms of APC are ineffective in forming APC/AXIN/GSK3/β-catenin complexes (9, 10, 11, 16),which phosphorylate β-catenin and lead to its degradation by the ubiquitin-proteasome system (17, 18, 19, 20). The resulting accumulation of β-catenin allows it to complex with Tcf-4, creating a bipartite transcription complex that activates downstream growth-promoting genes (21, 22). Accordingly, colorectal cancers exhibit high levels of constitutive β-catenin/Tcf-4-mediated transcription, which can be suppressed by exogenous APC(23). Moreover, mutations of β-catenin that render it resistant to APC-mediated down-regulation have been identified in the unusual colorectal tumors that express wild-type APC(24) as well as in other tumor types(25, 26, 27, 28, 29, 30, 31). Finally, recent studies have identified direct downstream targets of CRT, including the growth promoting genes c-MYC and cyclin D1 (32, 33, 34, 35).

The findings reviewed above suggest that APC suppresses tumorigenesis by inhibiting CRT of growth-promoting genes and subsequently inducing apoptosis. However, direct evidence for this scenario is still lacking. In this study, we determined the ability of the β-catenin-binding domain of APC in isolation to inhibit CRT, down-regulate expression of growth-promoting target genes, and inhibit tumor cell growth in vitro and in vivo.

Cell Culture, Medium, and Reagents.

The 293 cell line was purchased from Microbix Biosystems (Toronto,Canada), and 911 cells were kindly provided by Alex J. Van der Eb of the University of Leiden. Both lines were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml of penicillin, and 100 μg/ml of streptomycin. The human colon carcinoma cell lines, SW480, DLD1,HCT116, LoVo, SW48, HT29, SW837, and SW1417, were obtained from the American Type Culture Collection and cultivated in McCoy’s 5A media(Life Technologies, Inc., Gaithersburg, MD) supplemented with 10%fetal bovine serum (HyClone), 100 units/ml of penicillin, and 100μg/ml of streptomycin. The DOT and Dluc cell lines were derived from DLD1 cells as described below. Both cell lines were maintained in McCoy’s 5A media (Life Technologies) supplemented with 10% fetal bovine serum (HyClone) and 0.4 mg/ml Hygromycin B (Calbiochem).

Generation of Recombinant Adenovirus Expressing cAPC (Ad-CBR).

The recombinant adenovirus, Ad-CBR, which carried the cAPC, was generated using a modified system as previously described(36). The cAPC containing amino acids 958-2075(nucleotides 2890–6240) was isolated from pCMV-APC by BglII digestion. This fragment was subcloned into the pEGFP-C1 (Clontech,Palo Alto, CA). The cassette containing the EGFP-tagged cAPC was further subcloned into the shuttle vector (pShuttle) using Apal I and Mlu I sites. Recombinant adenoviral plasmid was generated by homologous recombination in Escherichia. coli (BJ5183). BJ5183 cells were transformed using electroporation with pAdEasy-1 and pShuttle/EGFP-cAPC linearized with PmeI. Successful recombinants were identified by restriction endonuclease mapping. The recombinant EGFP-cAPC virus (Ad-CBR) was produced in the 911 and 293 adenovirus packaging lines, and the viral particles were purified by CsCl banding. The control virus (Ad-EGFP) with EGFP alone was also prepared and purified side by side. Viral titer was determined by a modified CPE end point assay. A series of Ad-GFP infections was performed on HCT116 cells to determine the optimal MOI to avoid adenovirus-associated CPE. Typically, viral CPE could be observed at an MOI of >100, which resulted in >80% of cells becoming fluorescent 18 h after Ad-GFP infection. To avoid any CPEs of viruses, we infected cells with a minimal MOI, generating fluorescence in 20–30% of the cells(MOI = 5–11), then flow-sorted infected cells to obtain homogeneous populations.

Viral Infection and Cell Sorting.

Viral stocks were predialyzed using 1% agarose in microcentrifuge tubes. Three million cells were infected with either Ad-CBR or Ad-GFP in a 75-cm2 flask. After 18 h of incubation at 37°C, cells were washed, trypsinized, and subjected to fluorescence-activated cell sorting. Cells with green fluorescence were collected for experiments or were replated in culture flasks immediately after sorting.

Reporter Assay.

DOT and Dluc cells were generated from DLD1 cells by cotransfection of pTK-hygro (Clontech) and a Tcf-4-responsive luciferase plasmid(pGL3-OT)4or a constitutive luciferase plasmid (pGL3-control; Promega, Madison,WI), respectively. Clones were isolated, and the sensitivity to CRT was determined using a dominant-negative Tcf-4 adenovirus.4 Luciferase reporter activity in the DOT clone was constitutively high as expected for a CRT-responsive reporter in a colorectal cancer cell line with mutated APC. This constitutive activity was inhibited by dominant-negative Tcf-4. In contrast, the luciferase activity in the Dluc clone was unaffected by dominant-negative Tcf-4 as expected for expression driven by the SV40 promoter. To assess the effects of cAPC on CRT, DOT and Dluc cells were infected with Ad-GFP and Ad-CBR. Eighteen h after viral infection, equal numbers of GFP-positive cells were pelleted, lysed,and collected for luciferase assays using luciferase assay reagents(Promega).

Western Blot Analysis.

Whole cell lysates were prepared in a solution containing 62.5 mm Tris-HCl (pH 6.8), 2% SDS, and 0.5%β-mecarptoethanol. Equal amounts of total protein from each lysate were loaded and separated on 4–12% Tris-Glycine-SDS polyacrylamide gels (Novex, San Diego, CA) and electroblotted to Millipore Immobilon-P polyvinylidene difluoride membranes. Western blots were developed by chemiluminescence (NEN Life Science, Boston, MA), detected by Kodak Image Station 440CF, and analyzed by one-dimensional Image Analysis software (NEN Life Science). Primary antibodies included anti-GFP polyclonal antibody from Clontech (Palo Alto, CA), anti-c-MYC monoclonal antibody (9E10) from Santa Cruz (Santa Cruz, CA), anticyclin D1 monoclonal antibody (A-12) from Santa Cruz, and anti-α-tubulin monoclonal antibody (TU-02) from Santa Cruz. Secondary peroxidase-conjugated antibodies were goat antimouse IgG and goat antirabbit IgG from Pierce (Rockford, IL).

Immunofluorescence Staining.

Cells were infected with Ad-GFP or Ad-CBR for 18 h and sorted. Fluorescent cells were cultured on an 8-well chamber CultureSlides(Becton Dickinson, Bedford, MA). After 8 h, cells were fixed in 3% paraformaldehyde in PBS at room temperature for 8 min, then permeabilized with 0.3% NP40 in PBS for another 8 min. After washing in PBS, the cells were incubated with primary mouse anti-β-catenin monoclonal antibody (1 μg/ml; Transduction Laboratories, Lexington,KY) at 4°C overnight. After washing, cells were incubated with biotinylated goat antimouse IgG (Pierce, Rockford, IL) at room temperature for 1 h. The immunoreactivity was revealed using Alexa568-conjugated streptavidin (Molecular Probes, Eugene, OR), and cells were counterstained with 10 μg/ml DAPI. The cells were examined under a Nikon fluorescence microscope (Image Systems, Columbia, MD).

Cell Growth and Colony Formation Assay.

Cells (105) were plated in one well of a 24-well plate. Cells were counted using a hemocytometer after trypsinization on days 1, 2, 3, and 5. For colony formation assays,each well of the 24-well plates was precoated with 100 μl of collagen gel containing 50% type I collagen (Collaborative Biomedical Science,Bedford, MA), 40% culture medium, and 0.75%NaHCO3 (Halttunen). One hundred sixty μl of collagen gel-cell suspension containing 10,000 cells, 45% type I collagen, 40% culture medium, and 0.075% NaHCO3were added to the wells. After solidification, each well was covered with 1 ml of culture medium, and the plates were incubated at 37°C. Twelve days after seeding, cells were stained with 0.05% crystal violet (Sigma, St. Louis, MO) containing 10% buffered formalin(Sigma).

DAPI Staining and Annexin V Staining for Apoptosis Detection.

Both attached and floating cells were harvested for staining. For DAPI staining, 3 × 105 cells were resuspended in 50 μl of PBS and 350 μl of staining solution containing 0.6% NP40, 3% paraformaldehyde, and 10 μg/ml DAPI. For annexin V staining, 105 cells were suspended in 100 μl of annexin-binding buffer containing 10 mm HEPES,140 mm NaCl, and 2.5 mmCaCl2. Five μl of Alexa568-conjugated annexin V(Molecular Probes) were added and incubated at room temperature for 15 min, at which point an additional 400 μl of annexin-binding buffer were added to each sample. Apoptotic cells were defined as those cells containing condensed and/or fragmented nuclei after DAPI staining or were fluorescent after annexin V staining. At least 500 cells were counted, and the results were expressed as the percentage of apoptotic cells in each sample.

Characterization of the Ad-CBR Recombinant Adenovirus.

As noted above, understanding the structure-function relationships of APC has been hindered by the inability to readily restore APC tumor suppressor activity to human cells. To address this problem, we developed an adenovirus system that would allow the relatively facile evaluation of APC effects in a variety of cell lines. Three features of the adenovirus construction are of particular interest. First, to facilitate the actual construction of the adenoviral vectors, we used the AdEasy adenovirus system (36), which employs recombination in bacteria rather than in mammalian cells to generate recombinant adenovirus. Second, we included a GFP marker to allow easy identification of APC-expressing cells. This eliminates problems related to differences in infectivity and allows the use of viral titers well below levels that result in virus-induced CPE. Avoiding such nonspecific CPE is especially important for assessing tumor suppressive effects. Finally, we generated a virus that expressed a fusion protein (GFP/cAPC) containing GFP fused to the cAPC (Fig. 1 A). The employment of a fusion protein ensured that expression of GFP was coupled with APC and allowed positive verification of cAPC expression. The growth suppressive effects of tumor suppressor genes impose a powerful negative selection force that can result in loss of expression of the tumor suppressor gene even in the presence of a positive selection marker.

The central portion of APC was chosen for the following experiments because it contains all of the known β-catenin (4, 5)and axin/conductin (9, 10, 11) binding domains (Fig. 1,A) and is sufficient for promoting β-catenin degradation(17). Infection of SW480 cells with adenovirus-expressing GFP/cAPC (Ad-CBR) resulted in the production of a fusion protein of the expected size (150-kDa fusion) as determined by Western blot analysis with an anti-GFP antibody (Fig. 1,B). Fluorescence microscopy revealed that the GFP/cAPC fusion protein was diffusely present in the cytoplasm in all of eight colorectal cancer cell lines tested (Table 1 and examples in Fig. 1 C).

Ad-CBR Inhibits Tcf4/β-Catenin-mediated Transactivation.

One of the best-characterized functions of APC is its ability to inhibit CRT. To investigate the effects of GFP/cAPC on this function,we generated DLD1 cell lines with either an integrated Tcf-4-responsive luciferase reporter (DOT cells) or a reporter driven by the SV40 promoter (Dluc cells). Infection of DOT cells with Ad-CBR markedly suppressed luciferase activity, whereas a virus expressing GFP alone(Ad-GFP) had no effect (Fig. 2,A). This inhibition appeared to be specific because infection of Dluc cells with Ad-CBR had no effect on luciferase activity (Fig. 2,B). To determine whether the Ad-CBR suppression could be extended to endogenous targets of CRT, we evaluated the expression of c-MYC (32) and cyclin D1 (33), two recently described targets of the APC/β-catenin/Tcf-4 pathway. Expression of c-MYC and cyclin D1 was examined in three human colorectal cancer cell lines. In DLD1 and SW480, CRT was constitutively activated because of APC mutations (37), whereas in HCT116, CRT activation was due to a β-catenin mutation (24). Infection of SW480 and DLD1 cells with Ad-CBR resulted in a marked reduction of c-MYC protein levels as well as a reduction of cyclin D1 (Fig. 3). Ad-GFP infection had no inhibitory effect on either c-MYC or cyclin D1 protein levels in these cells. As expected, HCT116 cells, which possess a stabilizing β-catenin mutation, were relatively resistant to the effects of Ad-CBR (Fig. 3).

The subcellular localization of β-catenin was dramatically altered by Ad-CBR infection. Whereas mock-infected or Ad-GFP-infected DLD1 cells displayed predominantly nuclear β-catenin staining, cells infected with Ad-CBR showed cytoplasmic and membrane β-catenin staining with minimal nuclear staining (Fig. 4,A). A similar alteration in the subcellular distribution ofβ-catenin was observed in SW480 cells (Fig. 4 B). The selective depletion of nuclear β-catenin by Ad-CBR was consistent with the model for APC action proposed previously (17, 26). The nuclear β-catenin staining in HCT116 was significantly less striking and appeared similar in Ad-GFP- and Ad-CBR-infected cells (data not shown).

Ad-CBR Suppresses Cell Growth and Colony Formation in Colorectal Cell Lines.

In both DLD1 and SW480, Ad-CBR infection resulted in a clear growth inhibition by day 2 (Fig. 5,A). This growth inhibition was persistent, with Ad-CBR-infected DLD1 and SW480 cells failing to reach confluence even after 2 weeks of culture, long after mock- and Ad-GFP-infected cells became superconfluent. Consistent with the effects of Ad-CBR on CRT in HCT116 cells, the growth of Ad-CBR-infected HCT116 cells was only partially inhibited (Fig. 5 A).

We next examined the effects of Ad-CBR-mediated CRT inhibition on colony formation in collagen gels using a series of eight colorectal cancer cell lines (Table 1). The cells were flow-sorted to select virally infected cells, as described in “Materials and Methods.”Infection with Ad-CBR resulted in a marked suppression of colony formation in SW480, DLD1, LoVo, HT29, SW837, and SW1417 cells, all of which possess mutated APC (Fig. 5,B). Colony numbers were reduced by at least 96% in each of these lines compared with cells mock-infected or infected with Ad-GFP virus. Examination under phase-contrast microscopy revealed that most of the Ad-CBR-infected cells were pyknotic. A small number of growth-arrested single cells remained in the gels, and these continued to express APC as judged by fluorescence. In the few colonies that did form after Ad-CBR infection,APC did not appear to be expressed. In contrast, cells with intact APC but mutated β-catenin were able to form a significant number of colonies following Ad-CBR infection (HCT116 and SW48 in Fig. 5 B).

Ad-CBR Induces Apoptosis in Colorectal Cell Lines.

Ad-CBR-expressing cells revealed a gradual loss of the G1 peak and an accumulation of cells in the S and G2 phases of the cell cycle (data not shown). Five days after infection, all six colorectal cell lines with APC mutations demonstrated significantly increased apoptosis after Ad-CBR infection (Fig. 6). In line with the effects of Ad-CBR on CRT and growth, the mutantβ-catenin-containing cell lines, HCT116 and SW48, exhibited little increase in apoptosis in response to Ad-CBR infection. Time course studies revealed that the first morphological signs of apoptosis were not evident in DLD1 and SW480 cells until 72 h after plating (data not shown). The induction of apoptosis was confirmed by Annexin V,which has been shown to bind to phosphatidylserine exposed on the outer leaflet of apoptotic cell membranes (38, 39). The proportion of DLD1 and SW480 cells staining with Alexa568-labeled annexin V was in good agreement with the fraction of cells displaying morphological signs of apoptosis (>90% of annexin V-labeled cells displayed apoptotic nuclei). The induction of apoptosis by Ad-CBR was equally evident in DLD1 cells grown as xenografts (data not shown).

Although the ability of APC to suppress intestinal tumorigenesis has been known for several years, the sequences required for this inhibition have not been well defined. In this study, we define a minimal portion of APC that is sufficient for its growth-suppressive effects. Our results suggest that expression of the cAPC is sufficient to inhibit cellular proliferation and induces apoptosis in colorectal cancer cells.

The biological effects of the cAPC are likely related to abrogation of the APC/β-catenin/Tcf-4 signaling pathway. This conclusion is based on the fact that Ad-CBR inhibits β-catenin nuclear translocation(Fig. 4), suppresses β-catenin/Tcf-4-mediated transcription in reporter assays (Fig. 2), and down-regulates the expression of targets of the APC/β-catenin/Tcf-4 pathway (Fig. 3). Cellular proliferation and colony formation are dramatically suppressed by Ad-CBR in cell lines that contain mutations in the APC gene, but are only partially inhibited in lines containing mutations of β-catenin that render it resistant to APC degradation.

At the cellular level, expression of the cAPC eventually results in apoptosis of colorectal cancer cells containing APCmutations. This observation is consistent with those in a previous report, demonstrating apoptosis 60 h after induction of full-length APC expression (2). In both cases, the delay in appearance of apoptotic cells suggests that APC-induced apoptosis may not be a direct result of suppression of CRT. It should also be noted that the above cited observations and those reported here were made with tumor human cell lines maintained in culture and that additional experiments will be necessary to confirm that they accurately reflect the growth effects of APC in primary human tumors.

Although the β-catenin-binding domain in the cAPC is sufficient for growth suppression by APC, it may not recapitulate all of the functions of this gene. For example, the carboxyl-terminal third of APC can associate with the human homologue of the Drosophila tumor suppressor gene discs large (hDLG; Ref. 15) and EB-1(12). The latter has recently been implicated in the spindle checkpoint (40, 41). In addition, a carboxyl-terminal fragment of APC has been shown to induce assembly and bundling of microtubules in vitro and has a role in directed cell migration (13, 14, 42). Like other canonical tumor suppressor genes, it is likely that APC functions at several levels to regulate cell growth and suppress neoplastic transformation. However,the finding that the middle third of APC is sufficient to inhibit tumor cell growth focuses further attention on the APC/β-catenin interaction. Future experiments to understand the upstream regulators and downstream transducers of this interaction should shed further light on tumorigenesis associated with defects in the APC pathway.

Fig. 1.

Characterization of the Ad-CBR. A, map of GFP/cAPC incorporated into Ad-CBR. Linear representation of different domains in APC, including oligomerization domains, armadillo repeats,15-amino acid repeats, 20-amino acid repeats, SAMP repeats, the basic domain, and the EB-1 binding site, are shown. Theβ-catenin-binding and degradation domain, which comprises 15-amino acid repeats, 20-amino acid repeats, and SAMP repeats, was fused with the carboxyl-terminal of GFP. Expression of this cassette is driven by a cytomegalovirus promoter in the Ad-CBR adenovirus vector. B, Western blot analysis with an anti-GFP antibody. Infection of SW480 cells with Ad-CBR resulted in production of a fusion protein of the expected size (150 kDa), whereas the Ad-GFP-infected cells generated the expected 17-kDa GFP. C, fluorescence microscopy revealed that the GFP/cAPC fusion protein was diffusely localized to the cytoplasm in DLD1 and HCT116 cells.

Fig. 1.

Characterization of the Ad-CBR. A, map of GFP/cAPC incorporated into Ad-CBR. Linear representation of different domains in APC, including oligomerization domains, armadillo repeats,15-amino acid repeats, 20-amino acid repeats, SAMP repeats, the basic domain, and the EB-1 binding site, are shown. Theβ-catenin-binding and degradation domain, which comprises 15-amino acid repeats, 20-amino acid repeats, and SAMP repeats, was fused with the carboxyl-terminal of GFP. Expression of this cassette is driven by a cytomegalovirus promoter in the Ad-CBR adenovirus vector. B, Western blot analysis with an anti-GFP antibody. Infection of SW480 cells with Ad-CBR resulted in production of a fusion protein of the expected size (150 kDa), whereas the Ad-GFP-infected cells generated the expected 17-kDa GFP. C, fluorescence microscopy revealed that the GFP/cAPC fusion protein was diffusely localized to the cytoplasm in DLD1 and HCT116 cells.

Close modal
Fig. 2.

Ad-CBR suppresses CRT. A, the luciferase activity was dramatically inhibited in Ad-CBR-infected DOT cells as compared with control and Ad-GFP-infected DOT cells. B,no significant differences in luciferase activity among control,Ad-GFP-infected, and Ad-CBR-infected Dluc cells were observed at days 1 and 2. Values are the average of three experiments.

Fig. 2.

Ad-CBR suppresses CRT. A, the luciferase activity was dramatically inhibited in Ad-CBR-infected DOT cells as compared with control and Ad-GFP-infected DOT cells. B,no significant differences in luciferase activity among control,Ad-GFP-infected, and Ad-CBR-infected Dluc cells were observed at days 1 and 2. Values are the average of three experiments.

Close modal
Fig. 3.

Western blot analysis of c-MYC and cyclin D1 in DLD1,SW480, and HCT116. As compared with the control and Ad-GFP-infected cells, c-MYC expression was strongly repressed in both Ad-CBR-infected DLD1 and SW480 cells but is only partially inhibited in Ad-CBR-infected HCT116. Similarly, repression of cyclin D1 expression by Ad-CBR is observed in DLD1and SW480 but not in HCT116. Similar expression levels of α-tubulin in each lane indicate that similar amounts of protein are loaded.

Fig. 3.

Western blot analysis of c-MYC and cyclin D1 in DLD1,SW480, and HCT116. As compared with the control and Ad-GFP-infected cells, c-MYC expression was strongly repressed in both Ad-CBR-infected DLD1 and SW480 cells but is only partially inhibited in Ad-CBR-infected HCT116. Similarly, repression of cyclin D1 expression by Ad-CBR is observed in DLD1and SW480 but not in HCT116. Similar expression levels of α-tubulin in each lane indicate that similar amounts of protein are loaded.

Close modal
Fig. 4.

Immunofluorescence staining of β-catenin in DLD1(A) and SW480 (B). Cells were infected with Ad-CBR or Ad-GFP as indicated. Cells were stained for β-catenin and counterstained with DAPI as indicated. Ad-GFP-infected cells demonstrate a predominant nuclear staining of β-catenin. In contrast,almost all of the Ad-CBR-infected cells exhibited cytoplasmic and membrane β-catenin staining with minimal nuclear staining.

Fig. 4.

Immunofluorescence staining of β-catenin in DLD1(A) and SW480 (B). Cells were infected with Ad-CBR or Ad-GFP as indicated. Cells were stained for β-catenin and counterstained with DAPI as indicated. Ad-GFP-infected cells demonstrate a predominant nuclear staining of β-catenin. In contrast,almost all of the Ad-CBR-infected cells exhibited cytoplasmic and membrane β-catenin staining with minimal nuclear staining.

Close modal
Fig. 5.

Cell growth and colony formation assays. A,growth kinetics in DLD1, SW480, and HCT116 cells after mock, Ad-GFP, or Ad-CBR infections, as indicated. B, colony formation in collagen gel after mock, Ad-GFP, or Ad-CBR infections, as indicated. The number of colonies is indicated below each well.

Fig. 5.

Cell growth and colony formation assays. A,growth kinetics in DLD1, SW480, and HCT116 cells after mock, Ad-GFP, or Ad-CBR infections, as indicated. B, colony formation in collagen gel after mock, Ad-GFP, or Ad-CBR infections, as indicated. The number of colonies is indicated below each well.

Close modal
Fig. 6.

Apoptosis in virus-infected cells. Cells were stained with DAPI dye, and at least 500 cells were counted. The results are expressed as the fold increase in the percentage of apoptotic cells in each sample.

Fig. 6.

Apoptosis in virus-infected cells. Cells were stained with DAPI dye, and at least 500 cells were counted. The results are expressed as the fold increase in the percentage of apoptotic cells in each sample.

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 NIH Grant CA57345 and Genzyme Molecular Oncology (Genzyme; to K. W. K.). B. V. and K. W. K. are consultants to Genzyme. B. V. is an investigator of the Howard Hughes Medical Institutes.

3

The abbreviations used are: APC, adenomatous polyposis coli; Tcf, T-cell factor; DAPI,4′,6-diamidino-2-phenylindole; cAPC, central third of APC; CBR,catenin-binding region; CRT, catenin-regulated transcription; GFP,green fluorescent protein; EGFP, enhanced GFP; CPE, cytopathic effect;MOI, multiplicity of infection.

4

J. Yu, unpublished data.

Table 1

Colorectal cancer cell lines

Colorectal cancer cell lines
DLD1HCT116SW480LOVOSW48SW1417HT29SW837
Microsatellite instability Yes Yes No Yes Yes No No No 
APC mutation Yes No Yes Yes No Yes Yes Yes 
β-catenin regulatory domain mutation No Yes No No Yes No No No 
Colorectal cancer cell lines
DLD1HCT116SW480LOVOSW48SW1417HT29SW837
Microsatellite instability Yes Yes No Yes Yes No No No 
APC mutation Yes No Yes Yes No Yes Yes Yes 
β-catenin regulatory domain mutation No Yes No No Yes No No No 

We thank members of the Johns Hopkins Oncology Center’s Molecular Genetics Laboratory for their helpful comments and discussion.

1
Kinzler K. W., Vogelstein B. Lessons from hereditary colon cancer.
Cell
,
87
:
159
-170,  
1996
.
2
Morin P. J., Vogelstein B., Kinzler K. W. Apoptosis, and APC in colorectal tumorigenesis.
Proc. Natl. Acad. Sci. USA
,
93
:
7950
-7954,  
1996
.
3
Groden J., Joslyn G., Samowitz W., Jones D., Bhattacharyya N., Spirio L., Thliveris A., Robertson M., Egan S., Meuth M., White R. Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.
Cancer Res.
,
55
:
1531
-1539,  
1995
.
4
Rubinfeld B., Souza B., Albert I., Muller O., Chamberlain S. H., Masiarz F. R., Munemitsu S., Polakis P. Association of the APC gene product with beta-catenin.
Science (Washington DC)
,
262
:
1731
-1734,  
1993
.
5
Su L. K., Vogelstein B., Kinzler K. W. Association of the APC tumor suppressor protein with catenins.
Science (Washington DC)
,
262
:
1734
-1737,  
1993
.
6
Hulsken J., Birchmeier W., Behrens J. E-cadherin and APC compete for the interaction with β-catenin and the cytoskeleton.
J. Cell Biol.
,
127
:
2061
-2069,  
1994
.
7
Rubinfeld B., Souza B., Albert I., Munemitsu S., Polakis P. The APC protein and E-cadherin form similar but independent complexes with alpha-catenin, beta-catenin, and plakoglobin.
J. Biol. Chem.
,
270
:
5549
-5555,  
1995
.
8
Rubinfeld B., Albert I., Porfiri E., Fiol C., Munemitsu S., Polakis P. Binding of GSK3-β to the APC-β-catenin complex and regulation of complex assembly.
Science (Washington DC)
,
272
:
1023
-1025,  
1996
.
9
Behrens J., Jerchow B. A., Wurtele M., Grimm J., Asbrand C., Wirtz R., Kuhl M., Wedlich D., Birchmeier W. Functional interaction of an axin homolog, conductin, with β-catenin, APC, and GSK3β.
Science (Washington DC)
,
280
:
596
-599,  
1998
.
10
Kishida S., Yamamoto H., Ikeda S., Kishida M., Sakamoto I., Koyama S., Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin.
J. Biol. Chem.
,
273
:
10823
-10826,  
1998
.
11
Hart M. J., de los Santos R., Albert I. N., Rubinfeld B., Polakis P. Down-regulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK3 β.
Curr. Biol.
,
8
:
573
-581,  
1998
.
12
Su L. K., Burrell M., Hill D. E., Gyuris J., Brent R., Wiltshire R., Trent J., Vogelstein B., Kinzler K. W. APC binds to the novel protein EB1.
Cancer Res.
,
55
:
2972
-2977,  
1995
.
13
Munemitsu S., Souza B., Muller O., Albert I., Rubinfeld B., Polakis P. The APC gene product associates with microtubules in vivo and promotes their assembly in vitro.
Cancer Res.
,
54
:
3676
-3681,  
1994
.
14
Smith K. J., Levy D. B., Maupin P., Pollard T. D., Vogelstein B., Kinzler K. W. Wild-type but not mutant APC associates with the microtubule cytoskeleton.
Cancer Res.
,
54
:
3672
-3675,  
1994
.
15
Matsumine A., Ogai A., Senda T., Okumura N., Satoh K., Baeg G-H., Kawahara T., Kobayashi S., Okada M., Toyoshima K., Akiyama T. Binding of APC to the human homolog of the drosophila discs large tumor suppressor protein.
Science (Washington DC)
,
272
:
1020
-1023,  
1996
.
16
Ikeda S., Kishida S., Yamamoto H., Murai H., Koyama S., Kikuchi A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin.
EMBO J.
,
17
:
1371
-1384,  
1998
.
17
Munemitsu S., Albert I., Souza B., Rubinfeld B., Polakis P. Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.
Proc. Natl. Acad. Sci. USA
,
92
:
3046
-3050,  
1995
.
18
Yost C., Torres M., Miller J. R., Huang E., Kimelman D., Moon R. T. The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.
Genes Dev.
,
10
:
1443
-1454,  
1996
.
19
Salomon D., Sacco P. A., Roy S. G., Simcha I., Johnson K. R., Wheelock M. J., Ben-Ze’ev A. Regulation of β-catenin levels and localization by overexpression of plakoglobin and inhibition of the ubiquitin-proteasome system.
J. Cell Biol.
,
139
:
1325
-1335,  
1997
.
20
Aberle H., Bauer A., Stappert J., Kispert A., Kemler R. β-catenin is a target for the ubiquitin-proteasome pathway.
EMBO J.
,
16
:
3797
-3804,  
1997
.
21
Behrens J., von Kries J. P., Kuhl M., Bruhn L., Wedlich D., Grosschedl R., Birchmeier W. Functional interaction of β-catenin with the transcription factor LEF-1.
Nature (Lond.)
,
382
:
638
-642,  
1996
.
22
Molenaar M., van de Wetering M., Oosterwegel M., Peterson-Maduro J., Godsave S., Korinek V., Roose J., Destree O., Clevers H. XTcf-3 Transcription Factor mediates β-catenin-induced axis formation in xenopus embryos.
Cell
,
86
:
391
-399,  
1996
.
23
Korinek V., Barker N., Morin P. J., van Wichen D., de Weger R., Kinzler K. W., Vogelstein B., Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.
Science (Washington DC)
,
275
:
1784
-1787,  
1997
.
24
Morin P. J., Sparks A. B., Korinek V., Barker N., Clevers H., Vogelstein B., Kinzler K. W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC.
Science
,
275
:
1787
-1790,  
1997
.
25
Robbins P. F., El-Gamil M., Li Y. F., Kawakami Y., Loftus D., Appella E., Rosenberg S. A. A mutated β-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes.
J. Exp. Med.
,
183
:
1185
-1192,  
1996
.
26
Rubinfeld B., Robbins P., El-Gamil M., Albert I., Porfiri E., Polakis P. Stabilization of β-catenin by genetic defects in melanoma cell lines.
Science (Washington DC)
,
275
:
1790
-1792,  
1997
.
27
de La Coste A., Romagnolo B., Billuart P., Renard C. A., Buendia M. A., Soubrane O., Fabre M., Chelly J., Beldjord C., Kahn A., Perret C. Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas.
Proc. Natl. Acad. Sci. USA
,
95
:
8847
-8851,  
1998
.
28
Voeller H. J., Truica C. I., Gelmann E. P. β-catenin mutations in human prostate cancer.
Cancer Res.
,
58
:
2520
-2523,  
1998
.
29
Miyoshi Y., Iwao K., Nagasawa Y., Aihara T., Sasaki Y., Imaoka S., Murata M., Shimano T., Nakamura Y. Activation of the β-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3.
Cancer Res.
,
58
:
2524
-2527,  
1998
.
30
Palacios J., Gamallo C. Mutations in the β-catenin gene (CTNNB1) in endometrioid ovarian carcinomas.
Cancer Res.
,
58
:
1344
-1347,  
1998
.
31
Zurawel R. H., Chiappa S. A., Allen C., Raffel C. Sporadic medulloblastomas contain oncogenic β-catenin mutations.
Cancer Res.
,
58
:
896
-899,  
1998
.
32
He T. C., Sparks A. B., Rago C., Hermeking H., Zawel L., da Costa L. T., Morin P. J., Vogelstein B., Kinzler K. W. Identification of c-MYC as a target of the APC pathway.
Science (Washington DC)
,
281
:
1509
-1512,  
1998
.
33
Tetsu O., McCormick F. β-catenin regulates expression of cyclin D1 in colon carcinoma cells.
Nature (Lond.)
,
398
:
422
-426,  
1999
.
34
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
.
35
Mann B., Gelos M., Siedow A., Hanski M. L., Gratchev A., Ilyas M., Bodmer W. F., Moyer M. P., Riecken E. O., Buhr H. J., Hanski C. Target genes of β-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.
Proc. Natl. Acad. Sci. USA
,
96
:
1603
-1608,  
1999
.
36
He T. C., Zhou S., da Costa L. T., Yu J., Kinzler K. W., Vogelstein B. A simplified system for generating recombinant adenoviruses.
Proc. Natl. Acad. Sci. USA
,
95
:
2509
-2514,  
1998
.
37
Smith K. J., Johnson K. A., Bryan T. M., Hill D. E., Markowitz S., Willson J. K., Paraskeva C., Petersen G. M., Hamilton S. R., Vogelstein B., Kinzler K. W. The APC gene product in normal and tumor cells.
Proc. Natl. Acad. Sci. USA
,
90
:
2846
-2850,  
1993
.
38
Vermes I., Haanen C., Steffens-Nakken H., Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V..
J. Immunol. Methods
,
184
:
39
-51,  
1995
.
39
Martin S. J., Reutelingsperger C. P., McGahon A. J., Rader J. A., van Schie R. C., LaFace D. M., Green D. R. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.
J. Exp. Med.
,
182
:
1545
-1556,  
1995
.
40
Muhua L., Adames N. R., Murphy M. D., Shields C. R., Cooper J. A. A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein.
Nature (Lond.)
,
393
:
487
-491,  
1998
.
41
Berrueta L., Kraeft S. K., Tirnauer J. S., Schuyler S. C., Chen L. B., Hill D. E., Pellman D., Bierer B. E. The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules.
Proc. Natl. Acad. Sci. USA
,
95
:
10596
-10601,  
1998
.
42
Nathke I. S., Adams C. L., Polakis P., Sellin J. H., Nelson W. J. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration.
J. Cell Biol.
,
134
:
165
-179,  
1996
.