The majority of colonic neoplasias contain mutations in either the adenomatous polyposis coli or the β-catenin (β-cat) gene, both of which result in elevated levels of cytoplasmic β-cat. The oncogenic activity of β-cat has been explored in vivo and in vitro with conflicting results. We tested the hypothesis that β-cat is capable of immortalizing and transforming cultured epithelial cells that represent the precursors to colon cancer. A truncated form of β-cat (ΔN89) was stably expressed in murine colonic epithelial cells that were conditionally immortalized by temperature-sensitive T antigen expression and contained a mutant ApcMin allele [Immorto-Min colonic epithelium (IMCE)]. IMCE cells, grown under nonpermissive conditions, were immortalized by expression of the truncated β-cat protein as determined by sustained growth in culture and escape from senescence as measured by endogenous β-galactosidase activity. IMCE neo cells at nonpermissive conditions underwent extensive apoptosis, an effect that was blocked by the expression of ΔN89β-catenin. IMCE β-cat cells had significantly lower p19 and p53 protein levels compared to IMCE neo cells, suggesting that alterations in these two key genes may mediate the effects of β-cat on both cellular senescence and apoptosis. IMCE β-cat cells were also transformed as determined by growth in the absence of serum, anchorage-independent growth, and sustained tumor growth in nude mice. Stable β-cat-expressing populations could not be generated in conditionally immortalized colonic epithelial cells with a wild-type Apc background. These studies demonstrated the immortalizing activity of stabilized β-cat for the first time and extend the transforming ability of mutated β-cat to a cell line representing a precursor to colorectal cancer.
β-Catenin (β-cat)3 is a multifunctional protein involved in a variety of cellular processes including cell-cell adhesion and intracellular signaling. β-cat, a component of the adherens junctions, binds to E-cadherin and links it to the actin cytoskeleton through its interaction with α-catenin (1, 2, 3) or vinculin (4). β-cat also plays a role in cell signaling by participation in the wnt/wingless pathway (reviewed in Ref. 5). When not complexed with E-cadherin at the cell junctions, β-cat can be found in a large complex with glycogen synthase kinase 3β (GSK-3β), conductin/axin, and adenomatous polyposis coli (APC). This complex down-regulates the levels of β-cat in conjunction with the ubiquitin-proteasome complex. Briefly, GSK-3β and β-cat complex with conductin/axin and APC in an arrangement where GSK-3β can phosphorylate serine/threonine residues in the NH2 terminus of β-cat. The phosphorylation of these residues is a critical step preceding ubiquitination and subsequent proteolytic degradation of β-cat by the ubiquitin-proteasome system (6). Recent data suggest that GSK-3β may be critical in facilitating the assembly of the complex containing β-cat, APC, and axin/conductin by phosphorylating other proteins in the complex (7, 8, 9).
In the event that β-cat is not degraded, it accumulates in the cytoplasm and translocates to the nucleus where it can bind to a family of DNA-binding proteins known as T-cell factor (Tcf) or lymphoid-enhancing factor (Lef) (10, 11, 12). β-cat-Tcf complex has been shown to be sufficient to activate transcription of artificial Tcf site promoters (12, 13). Recently, significant progress has been made in elucidating the target genes activated by β-cat. The proto-oncogene c-myc (14), the cell cycle regulator cyclin D1 (15), the matrix metalloproteinase matrilysin (16), and a number of additional genes (17, 18, 19, 20) have been identified as targets of the β-cat-signaling pathway.
Strong correlations exist between APC/β-cat mutations and colon cancer which suggest a causative role for β-cat in tumorigenesis. Truncating mutations in APC have been found in >70% of colon tumors examined (21). Genetic predisposition to intestinal cancer is characterized by a germ-line mutation in the APC gene in familial adenomatous polyposis (FAP) in humans (22, 23, 24). Individuals carrying a truncating mutation in the APC gene lose the remaining wild-type allele, resulting in loss of normal APC function in the benign tumors that develop (25, 26, 27, 28). The multiple intestinal neoplasia (Min) mouse mimics FAP in that these mice carry a mutation in one allele of Apc and lose the remaining wild-type allele in the tumors that develop (27, 29). The mutant APC is defective in its ability to promote proteolytic degradation of β-cat and leads to an activation of β-cat/Tcf-mediated transcription (12, 13). Alternatively, in some colon cancers, mutations can be found in β-cat itself that result in an equivalent effect (30, 31). These mutations occur in specific NH2-terminal serine/threonine residues that are normally phosphorylated by GSK-3β and thus prevent the phosphorylation of β-cat and its subsequent degradation (13). Thus, stabilization and activation of the β-cat/Tcf-signaling pathway is believed to be one mechanism by which APC exerts a gatekeeper function in the development of colon cancer.
The oncogenic activity of β-cat has been explored in vivo and in vitro with conflicting results. An NH2-terminal truncated form of β-cat (ΔN89) targeted to the skin of mice resulted in the development of structures that resembled differentiated tumors of the hair follicles (32). It has been previously demonstrated that expression of the NH2-terminal truncated β-cat (ΔN131) in the intestine of transgenic mice was sufficient to result in focal and infrequent adenoma formation (33). Alternatively, it has been reported that targeting ΔN89β-catenin to the intestine was not sufficient to cause adenoma formation (34). Although divergent effects of different truncating mutations of β-cat cannot be excluded as a reason for the opposing results in vivo, there has also been conflicting evidence on the ability of β-cat to transform cells in culture. It has been shown previously that β-cat possesses transforming ability in a rodent fibroblast cell, NIH 3T3 (35). However, Kolligs et al. (36) reported that overexpression of either wild-type or NH2-truncated β-cat failed to induce transformation in NIH 3T3 cells. Kolligs et al. (36) demonstrated that missense mutations of β-cat at serine 33 (S33Y) or in-frame deletion of regions in the NH2 terminus important for its degradation could induce neoplastic transformation of RK3E cells, an adenovirus immortalized epithelial cell line derived from neonatal rat kidney. However, the same β-cat mutations were not sufficient to transform the NIH 3T3 fibroblast cell line, the IEC 18 intestinal epithelial cell line, or the 1811 cells keratinocyte cell line (36). It has further been shown that β-cat mutations have different effects on signaling and transformation in canine kidney cells (Madin-Darby canine kidney cells) where deletion of the α-catenin binding site of β-cat was sufficient to induce anchorage-independent growth but shorter truncations were not (37). The reasons for these differences can be ascribed to both differential activities of different forms of mutant β-cat and to the different mechanism by which these cells previously had been immortalized such as adenovirus E1A protein.
The previous in vitro studies examined the ability of β-cat to transform previously immortalized, continuously growing cells in culture. However, in vivo APC or β-cat mutations exert their effect on normal epithelial cells. We were interested in testing the ability of β-cat to act at what is presumed to be the first step in the transformation process leading to colorectal cancer, i.e., to immortalize normal intestinal epithelial cells. To do so required a cell line that was not intrinsically immortal and expressed wild-type β-cat and APC. It has been previously shown by a number of groups that it is not possible to establish pure cultures of normal adult colonic epithelium (38, 39). To overcome this problem, we used conditionally immortalized cells isolated from the colon of the Immortomouse which express a temperature-sensitive SV40 large T antigen under the control of a IFN-γ-responsive promoter (40, 41). We report here that, under nonpermissive conditions, the immortomouse cells normally senesce and apoptose through a p19- and p53-mediated mechanism. However, when stable β-cat was expressed in these cells, they were protected from senescence and became immortal. Furthermore, β-cat expression resulted in full transformation of these cells as defined by anchorage-independent growth and tumor formation in susceptible animals.
MATERIALS AND METHODS
Cell Lines and Culture Conditions.
YAMC and IMCE cells were described previously (40) and were generously provided by Robert Whitehead (Ludwig Institute, Melbourne, Australia). Briefly, YAMC cells are normal murine colonic epithelial cells derived from the Immortomouse and are conditionally immortalized by a temperature-sensitive variant of the SV40 T antigen (tsA58) under the control of the H-2kb promoter (42). The expression of the T antigen is enhanced by an IFN-γ-responsive element in the H-2kb promoter. IMCE cells are mouse colon epithelial cells from an F1 Immorto/Min mouse that contain one mutant allele of Apc (ApcMin) and are also conditionally immortalized by the temperature-sensitive IFN-γ-responsive T antigen. YAMC and IMCE cells are immortal under permissive conditions of 33°C in the presence of 2.5 units/ml IFN-γ (Genzyme Diagnostics, Cambridge, MA), but are no longer viable upon removal of IFN-γ and a switch to 37°C (nonpermissive conditions). All cells were maintained at 33°C as a monolayer in DMEM (Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 2.5 units/ml IFN-γ, and 50 μg/ml gentamicin (Life Technologies).
Transfection of IMCE Cells.
Myc-tagged ΔN89β-catenin driven by the cytomegalovirus promoter in the pCAN vector containing neomycin resistance has been described previously (43). YAMC and IMCE cells were seeded at 70% confluency and transfection of the neomycin-resistance pCAN vector or the myc-ΔN89β-catenin construct was performed using Cellfectin (Life Technologies) according to the manufacturer’s instructions. Transfectants were selected under permissive conditions in medium supplemented with G418 (1 mg/ml; Mediatech Inc., Herndon, VA). Populations containing the neomycin-resistance vector are designated IMCE neo and populations containing the myc-tagged ΔN89β-catenin are designated IMCE β-cat. Pooled populations were maintained under permissive conditions in medium supplemented with 0.2 mg/ml G418.
Monolayer cultures were lysed using radioimmunoprecipitation buffer lysis [Tris-buffered saline (TBS), 5 mm Tris-HCl (pH 8) and 150 mm NaCl; 0.5% deoxycholate; 0.1% SDS; 1% NP40; 10 μg/ml aprotinin; 10 μg/ml leupeptin; and 1 mm phenylmethylsulfonyl fluoride; Sigma, St. Louis, MO] to generate total cell lysates. Loading of lanes was normalized to protein concentration as determined by the bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL). Electrophoresis was performed on 10% SDS-polyacrylamide gels and the protein was transferred to nitrocellulose membranes (Micron Separations Inc., Westborough, MA). The blots were blocked in a solution of TBS containing 5% nonfat dry milk. The following antibodies were diluted in TBS containing 0.5% Tween 20 (Sigma) and 5% nonfat dried milk: mouse monoclonal anti-myc antibody clone 9E10 (1:200; Calbiochem, La Jolla, CA), mouse anti-β-cat antibody clone 14 (1:1000; Transduction Laboratories, Lexington, KY), rabbit anti-cyclin D1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-p19 (0.5 μg/ml; Abcam, Cambridge, United Kingdom), and mouse anti-p53 antibody clone 421 (1:100; Oncogene Research Products, Boston, MA). The blots were incubated with a 1:10,000 dilution of biotinylated goat antimouse IgG (Vector Laboratories, Burlingame, CA) or goat antirabbit IgG (Vector Laboratories) followed by a 1:20,000 dilution of peroxidase-conjugated streptavadin (Jackson ImmunoResearch, West Grove, PA). The membranes were washed three times between each step for 10 min in TBS containing 0.5% Tween 20. Enhanced chemiluminescence was performed according to manufacturer’s instructions (Amersham, Piscataway, NJ) to detect antibody complexes.
Electrophoretic Mobility Shift Assay.
Nuclear lysates were prepared from IMCE neo and IMCE β-cat cells grown at permissive conditions and HT29 cells. EMSA was performed as described previously (16). Briefly, the +8-Tcf (5′-AACATCAAAGTAGCTCTGAGAA3-′) oligonucleotide was end labeled with polynucleotide kinase in the presence of [γ-32P]ATP. Labeled oligonucleotides (5 × 104 cpm) were incubated with 5 μg of crude nuclear extracts for 30 min at 37°C. For antibody competition, 250 ng of anti-β-cat (clone 6F9; Sigma) or nonspecific antibody (clone 9E10) was incubated in the binding reaction of HT29 cells. Samples were run on a 4% acrylamide/2% glycerol/0.25× Tris-borate EDTA gel and visualized by autoradiography.
IMCE neo and IMCE β-cat cells were trypsinized and plated at a density of 104 cells/35-mm dish. Every 2 days cells were trypsinized and viable cells were identified by trypan blue exclusion and counted using a hemocytometer.
Senescence of IMCE neo and IMCE β-cat cells was determined by staining the cells for senescence-associated β-galactosidase activity. Staining was performed as described previously (44). Briefly, 50,000 IMCE neo and IMCE β-cat cells were plated on Labtek chamber slides (Nunc, Naperville, IL) and grown at nonpermissive conditions. Cells were washed in PBS, fixed in 3% Formalin (Fisher Scientific, Pittsburgh, PA) for 5 min, washed in PBS, and incubated with senescence stain solution consisting of 1 mg of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Fisher Scientific) in dimethylformamide per ml of 40 mm C6H5O7Na3 (pH 6), 5 mm K4Fe(CN)6, 5 mm K3Fe(CN)6, 150 mm NaCl, and 2 mm MgCl2 for 16 h at 37°C. Twenty microscopic fields were counted for each cell type at the indicated time points.
Flow cytometry was performed as described previously (45). Briefly, 105 cells were seeded on 35-mm dishes and grown at permissive or nonpermissive conditions for 72 h. Cells were harvested by trypsinization, pelleted by centrifugation, washed three times in PBS [0.876 mm K2HPO4, 8.1 mm Na2HPO4 (pH 7.2), 137 mm NaCl, and 2.68 mm KCl] with 5 mm EDTA, and resuspended in 0.3 ml of the same buffer to which 0.7 ml of 100% ethanol was added. The cells were pelleted and resuspended in PBS with 50 μg/ml RNase A and incubated at room temperature for 30 min. Propidium iodide (Sigma) was added to the cells to a final concentration of 50 μg/ml to stain the DNA. Flow cytometry was performed on a FACStar machine (Becton Dickinson, San Jose, CA) and data analysis was performed with the Flow Jo data system (Treister Enterprises, San Diego, CA).
Hoechst staining was performed on 104 IMCE neo or IMCE β-cat cells plated on Labtek chamber slides and grown for 72 h at 37°C in the absence of IFN-γ (nonpermissive conditions). Cells were fixed in a solution of 3 parts methanol + 1 part glacial acetic acid, washed in PBS, and stained for 10 min with Hoechst stain (0.1 ng/ml bisbenzimide in PBS; Sigma). Nuclear morphology was visualized under UV fluorescence using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).
Soft Agar Assays.
Cells were trypsinized and single-cell suspensions of 10,000 cells were mixed with 0.3% agar (Difco, Detroit, MI) in DMEM plus 5% FBS and plated in 35-mm dishes over a 1-ml basal layer of 0.5% agar in DMEM plus 5% FBS. The cells were grown at permissive or nonpermissive conditions and 250 μl of medium were added to the plates every 4 days. After 14 days, the number of colonies was determined by counting the visible colonies under a dissecting microscope.
Cells (106/200 μl DMEM) were injected s.c. into 8-week-old female athymic nude (Harlan Sprague Dawley, Indianapolis, IN) mice. Tumor diameters were taken weekly by caliper rule measurement in two dimensions. Tumor volume was calculated using the formula
Establishment of Cell Lines Expressing ΔN89β-Catenin.
To determine whether β-cat has immortalizing capability, we used colonic epithelial cell lines that expressed functionally wild-type APC, wild-type β-cat, and were not immortal. YAMC cells are conditionally immortalized under permissive conditions by a temperature-sensitive SV40 large T antigen whose expression is enhanced by the addition of IFN-γ. YAMC cells were transfected with a cDNA encoding a truncated stable form of β-cat (ΔN89) under the control of the cytomegalovirus promoter (43). This protein lacks the first 89 amino acids of the NH2 terminus, which eliminates the critical serine and threonine residues in this region, blocking the degradation of β-cat and allowing for the accumulation of cytoplasmic pools that are free to move to the nucleus and activate transcription. Despite repeated attempts, we were unable to generate cells with stable expression of the ΔN89β-catenin protein in the YAMC cells. However, in parallel experiments, ΔN89β-catenin-expressing clones were obtained from IMCE cells derived from the colonic mucosa of an F1 Immortomouse-Min mouse hybrid (41). The IMCE cells contain one mutant ApcMin allele and one wild-type Apc allele and express both wild-type and truncated APC protein. Like YAMC cells, IMCE cells proliferate when maintained at permissive conditions (33°C in the presence of 2.5 units/ml IFN-γ), but when switched to the nonpermissive conditions (37°C in the absence of IFN-γ) the cells fail to proliferate and a high percentage of cells die (41). Populations of stable β-cat transfectants of IMCE cells were selected by culturing the cells in media containing G-418 under permissive conditions to avoid selecting for spontaneous immortalization. Pooled populations of cells referred to as IMCE neo, containing only the selection vector, and IMCE β-cat were generated. Expression of ΔN89β-catenin protein in the IMCE β-cat cells was confirmed by Western blot analysis using antibodies to the myc tag that was present in the transfected ΔN89β-catenin-expressed protein (Fig. 1,A) as well as antibodies to the β-cat protein (Fig. 1 B).
To determine whether the ΔN89β-catenin transfected into the IMCE cells was transcriptionally competent, we performed an EMSA using a consensus Tcf site as described previously (16). Nuclear extracts from IMCE β-cat cells recognized the radiolabeled oligonucleotide and resulted in a shift in the mobility of the bands, whereas nuclear extracts from IMCE neo cells did not produce the shifted band (Fig. 1,C). This band was confirmed to be β-cat by competition with an anti-β-cat antibody described previously (data not shown and Ref. 16). We analyzed the status of the Apc alleles in the IMCE β-cat cells using a PCR strategy described previously by Luongo et al. (29) to determine whether the cells retained the wild-type allele of Apc. The wild-type and Min alleles of Apc were present in these cells at roughly equivalent levels (data not shown), indicating that the presence of β-cat-Tcf complexes was a result of the transfected β-cat and not a loss of the wild-type Apc allele. In addition, the level of endogenous β-cat was unaltered in IMCE β-cat cells compared to IMCE neo cells (Fig. 1 B). These results indicate that the transfected ΔN89β-catenin is capable of complexing with Tcf/Lef and specific DNA sequences and therefore is likely to be transcriptionally active.
Stable β-cat Expression Induced Immortalization of IMCE Cells.
To determine whether β-cat expression is capable of immortalizing cells, control IMCE neo and IMCE β-cat cells were cultured under nonpermissive conditions and their growth was measured over a 6-day period. Cells expressing ΔN89β-catenin continued logarithmic growth under nonpermissive conditions, whereas the control IMCE neo cells were incapable of sustained growth at this temperature and in the absence of IFN-γ (Fig. 2 A). Thus, the expression of stabilized β-cat conferred the ability to survive and grow in culture to colonic epithelial cells.
Normal somatic cells have a finite life span and undergo a process of replicative senescence both in vitro and in vivo. Although rodent cells can often spontaneously immortalize by the selection of cells that escape crisis, the IMCE cells under nonpermissive conditions represent normal colonic epithelial cells that have not been selected for crisis survival. To determine whether IMCE β-cat cells have altered replicative senescence, IMCE neo and IMCE β-cat cells were assayed for β-galactosidase activity, a marker for cellular senescence (44). Cells were cultured in 5% FBS at 37°C without IFN-γ (nonpermissive conditions) and the percentage of cells expressing β-galactosidase activity at pH 6 was determined. Three days after the transfer to nonpermissive conditions, approximately 75% of IMCE neo cells were positive for β-galactosidase activity (Fig. 2, B and C). In contrast, no β-galactosidase-positive IMCE β-cat cells were detected at any time point examined. Thus, the expression of stabilized β-cat resulted in escape from cellular senescence, or immortalization, of IMCE cells.
β-cat Protects IMCE Cells from Apoptosis with Minimal Effects on Proliferation.
The IMCE β-cat cells were immortalized as shown by continued growth at nonpermissive conditions (Fig. 2,A). To determine whether β-cat expression influenced the rate of cell death, we examined the apoptotic index in the IMCE β-cat cells compared to the IMCE neo cells. Both cell types were cultured under permissive and nonpermissive conditions and the DNA was stained with propidium iodide. DNA profiles were examined by flow cytometry to determine the degree of DNA fragmentation characteristic of apoptotic cells. Converting IMCE neo cells from permissive to nonpermissive conditions resulted in an approximate 8-fold increase in the percentage of cells undergoing cell death as indicated by the percentage of DNA in the sub-G1 peak (Fig. 3,A). At nonpermissive conditions, approximately 30% of IMCE neo cells were apoptotic. In contrast, IMCE β-cat cells had a lower percentage of apoptotic cells at both permissive and nonpermissive conditions, so that at the nonpermissive conditions only 3.0% of the cells were apoptotic (Fig. 3,B). Thus, there was a 10-fold reduction in the apoptotic index at nonpermissive conditions in IMCE β-cat cells as compared to IMCE neo control cells. Apoptosis at nonpermissive conditions was confirmed by examination of nuclear morphology following Hoechst staining (Fig. 3, C and D).
The expression of β-cat has been reported to alter cell proliferation (46). To determine whether β-cat influenced the rate of growth of already immortalized colonic epithelial cells, growth curves were performed with IMCE cells immortalized with active T antigen, with ΔN89β-catenin, and with both active T antigen and ΔN89β-catenin. Expression of ΔN89β-catenin in IMCE cells resulted in a 1.4-fold (P < 0.05) greater rate of proliferation than active T antigen (Fig. 4,A, IMCE neo plus IFN-γ versus IMCE β-cat without IFN-γ). There was no additional effect on proliferation when both T antigen and β-cat were active (IMCE β-cat plus IFN-γ). Because the cell cycle regulator cyclin D1 has been shown to be a target of β-cat signaling (15) and ΔN89β-catenin is capable of complexing with a Tcf/Lef factor to bind to the consensus site (Fig. 1,C), we examined the levels of cyclin D1 by Western blot analysis in IMCE cells under the same conditions to determine whether the subtle difference in proliferative capacity correlates with changes in cyclin D1 levels. Densitometric analysis of cyclin D1 levels revealed that there was no significant difference in the level of expression of cyclin D1 in IMCE cells with active T antigen or with expression of ΔN89β-catenin (Fig. 4 B). Additionally, expression of both active T antigen and ΔN89β-catenin in IMCE cells did not result in increased levels of expression of cyclin D1 compared with either active T antigen or ΔN89β-catenin alone. Thus, ΔN89β-catenin was slightly more effective in promoting the growth of IMCE cells in vitro than T antigen but provided no significant proliferative advantage to previously immortalized IMCE cells.
β-cat Expressing IMCE Cells Have Reduced p19/ARF and p53.
IMCE β-cat cells undergo significantly less apoptosis and senescence than IMCE neo cells at nonpermissive conditions (Figs. 2 and 3). To explore the molecular basis of this difference, we examined p19/ARF and p53 levels in IMCE neo and IMCE β-cat cells at nonpermissive conditions. p19/ARF, a tumor suppressor encoded by the INK4/ARF locus, is up-regulated during senescence and disrupts the interaction of p53 and MDM2 by sequestering MDM2 to the nucleolus (47, 48). This disruption leads to the accumulation of p53 and subsequent cell cycle arrest in G1 and G2 or apoptosis (49, 50, 51, 52, 53, 54, 55). IMCE neo cells averaged 6-fold more p19 protein than IMCE β-cat cells at nonpermissive conditions (Fig. 5,A). p53 levels were on average 9-fold greater in the IMCE neo cells than in IMCE β-cat cells at nonpermissive conditions (Fig. 5,B). The appearance of degradation products of p53 was observed with the concomitant loss of full-length p53 in IMCE β-cat cells at nonpermissive conditions (Fig. 5 B). Taken together, these data suggest that β-cat expression blocked the induction of p19 at nonpermissive conditions and subsequent senescence and apoptosis mediated through p53.
Stabilized β-cat Transforms IMCE Cells.
Additional hallmarks of a transformed cell in vitro include the ability to grow in the presence of lowered concentrations of growth factors or serum and anchorage-independent growth. Previous work by Whitehead and Joseph (41) has shown that the parental IMCE cells are not capable of growth in culture in the absence of serum. However, IMCE cells expressing stabilized β-cat continued to proliferate in the absence of serum at nonpermissive conditions while the IMCE neo control cells did not (Fig. 6 A). Induction of T antigen in IMCE neo cells at the nonpermissive 37°C temperature by the addition of IFN-γ had no effect on the growth of the cells, suggesting that any up-regulatory effects of β-cat on the H-2Kb promoter that might occur were irrelevant (data not shown). These results confirmed that the effect of β-cat on the immortalization of the IMCE cells was independent of the resident T antigen gene.
IMCE neo and IMCE β-cat cells were cultured in soft agar to determine their requirement for anchorage-dependent growth. Under permissive conditions, IMCE β-cat cells formed 693.1 ± 94.9 colonies/35-mm dish while IMCE neo cells permitted the formation of 9.8 ± 2.9 colonies in soft agar (Fig. 6 B). Additionally, there was no difference in the number of colonies formed in the IMCE β-cat cells at permissive or nonpermissive conditions (data not shown). These data demonstrate that expression of stabilized β-cat is sufficient to permit growth in the absence of serum and to permit anchorage-independent growth, providing support for the hypothesis that β-cat can transform colonic epithelial cells.
The most definitive test of cellular transformation is the ability to confer growth in susceptible animals. We tested whether expression of the stabilized β-cat was sufficient to permit tumor formation in nude mice. It had previously been reported that the parental IMCE cells have a very low tumorigenic potential (41). IMCE neo and IMCE β-cat cells were injected s.c. into athymic nude mice and tumors were measured over a 3-month period. Injection of the cells expressing stabilized β-cat resulted in aggressive tumors such that by day 13 all mice injected with IMCE β-cat cells had visible tumors, whereas tumors were delayed or did not form in IMCE neo-injected mice (Fig. 6,C). IMCE β-cat-derived tumors were also an average of 5.8-fold larger in volume at 80 days postinjection compared to IMCE neo control cells (Fig. 6 D). These results demonstrate that ΔN89β-catenin-expressing cells have a high tumorigenic potential and support the hypothesis that β-cat can act as an oncogene by immortalizing and fully transforming colonic epithelial cells.
The use of Immortomouse-derived cells allowed us to explore the effects of stabilized β-cat on the cellular precursors of colorectal cancer in a culture system. We demonstrated for the first time that ΔN89β-catenin immortalized colonic epithelial cells. The IMCE cells mimic the normal colonic epithelium of a FAP patient; they exhibit normal cellular control mechanisms under nonpermissive conditions in a genetic background consisting of a mutation in a single allele of the Apc gene. Immortalization is a key step in the process of transformation and occurs through mis-regulation of pathways controlling cellular proliferation and apoptosis, resulting in life span extension. ΔN89β-catenin immortalized IMCE cells as demonstrated by the ability to undergo sustained growth in culture and escape from senescence, a property that is indicated by the production of endogenous β-galactosidase activity. In addition, ΔN89β-catenin prevented IMCE cells from undergoing apoptosis at nonpermissive conditions.
Immortalization involves several key proteins that function to alter the cell cycle and cell death signaling pathways. One of these key proteins is p19/ARF, a member of the INK4 family of cyclin D-dependent kinase inhibitors that plays a crucial role in blocking cell cycle progression. p19 functions to regulate the cell cycle and apoptosis by blocking turnover of the tumor suppressor p53. p53 is normally targeted for degradation by the ubiquitin-proteosome system by MDM2, an E3 ubiquitin ligase. p19 sequesters MDM2 and blocks the degradation of p53, which results in p53 accumulation. A number of p53 target genes have been shown to be involved in promoting cell cycle arrest and apoptosis. IMCE neo cells expressed both p19 and p53 at nonpermissive conditions, correlating with senescence and apoptosis in these cells. In contrast, IMCE β-cat cells expressed low levels of both p19 and p53 at nonpermissive conditions and did not undergo senescence or apoptosis at nonpermissive conditions. We have shown that β-cat associated with Tcf in nuclear extracts of the IMCE β-cat cells, suggesting that β-cat may regulate p19 expression directly. However, it is important to note that not all effects of β-cat on the transformed phenotype are related to its effects on transcription (37). At this time, the mechanism of p19 regulation has not been determined. Miyagishi et al. (56) demonstrated that β-cat can inhibit ectopically expressed p53 function by competing for transcriptional coactivators. However, in the IMCE cells we have shown that the effects of β-cat stabilization led to the down-regulation of the p53 protein itself.
The reduction in apoptosis observed in the IMCE β-cat cells compared to IMCE neo cells is consistent with a report by Orford et al. (57) that also demonstrated that β-cat significantly protected cells from apoptosis. However, Kim et al. (58) recently reported that overexpression of β-cat in various colon carcinoma cells induces apoptosis. Several factors may contribute to this discrepancy. Colon carcinoma cells contain genetic alterations not found in IMCE cells that may alter their responsiveness to β-cat. Also, IMCE β-cat cells were examined after a selection period whereas the colon carcinoma cells were tested for apoptosis immediately after introduction of β-cat. Finally, Kim et al. (58) overexpressed β-cat to a level greatly exceeding the already stable endogenous pool, whereas in our studies ectopic ΔN89β-catenin levels were roughly equivalent to the levels of normal, endogenous β-cat. Thus, based on the IMCE model, we conclude that the effect of β-cat stabilization is to allow colonic epithelial cells to escape from normal control mechanisms that limit cellular life span. Immortalization and a reduction in apoptosis can play a significant role in colorectal tumorigenesis in vivo, where increased survival could allow cells to accumulate additional mutations that enhance tumor progression (59).
Our studies also extend the cell types in which β-cat has been shown to have transforming activity to a colonic epithelial-derived cell line. IEC 18 cells, derived from rat intestinal epithelium, were not transformed by the S33Y-mutated β-cat or by β-cat with an in-frame deletion of the NH2 terminus of β-cat (36). In contrast, expression of ΔN89β-catenin transformed IMCE cells grown under nonpermissive conditions as determined by the ability of IMCE β-cat cells to grow in the absence of serum, to grow in an anchorage-independent environment, and to achieve sustained tumor growth in nude mice. It is not clear at this time if the difference is attributable to the different β-cat mutations used in these studies or differences in the properties of the epithelial-derived cell lines. However, since all of the β-cat mutations tested are associated with human colorectal cancer and the S33Y and in-frame deletion variants have been shown to transform RK3E adenovirus immortalized rat kidney cells (36), we suggest that the oncogenic activity of β-cat becomes apparent only in the context of complementary cellular alterations.
It is interesting that we were able to demonstrate the oncogenic effect of β-cat in IMCE cells, but not their wild-type YAMC equivalent. The only known difference between the two cell lines is the mutation of one allele of APC in the IMCE cells. It is possible that the partial loss of APC function results in an elevation in endogenous β-cat levels which, in combination with ectopically expressed ΔN89β-catenin, moves beyond a relevant threshold of β-cat to cause immortalization and transformation in the IMCE cells. Alternatively, it is also possible that mutant APC may cooperate with stabilized β-cat to allow immortalization and transformation. Previous work by D’abaco et al. (60) indicated that mutation of one allele of Apc was capable of synergizing with activated ras to transform the IMCE cells, whereas YAMC cells expressing only wild-type APC were not transformed by activated ras. The COOH terminus of APC, which is deleted in a high percentage of intestinal tumors, binds to a large number of proteins including the homologue of the Drosophila disc large tumor suppressor protein (DLG), EB1, microtubules, p34cdc2, and Asef (61, 62, 63, 64, 65, 66, 67, 68, 69). Additionally, APC has been shown to be involved in a number of different cellular processes including cell cycle regulation, cell migration, microtubule assembly, cell adhesion, apoptosis, and cell fate determination (61, 70, 71, 72, 73). Thus, deletion of the COOH terminus of one allele of Apc could result in alterations in the regulation of any number of cellular pathways and processes. These results help resolve the controversy surrounding the oncogenic potential of stabilized β-cat. For example, the RK3E epithelial cells in the study by Kolligs et al. (36) may have alterations in a relevant complementary pathway, whereas cells such as the IEC18 rat intestinal epithelial cells do not. Thus, similar to the complementary roles ascribed for oncogenes such as myc and ras (74), β-cat may transform normal colonic epithelial cells in conjunction with a pathway that is activated by the presence of a COOH-terminal truncated form of APC. Perhaps one reason that APC is a critical gatekeeper gene for colon cancer is that mutations in APC can lead to the activation of two pathways that cooperate to immortalize and transform intestinal epithelial cells.
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Supported by NIH Grant CA 60867. R. A. W. is a predoctoral fellow supported by NIH Grant T32 CA 09385.
The abbreviations used are: β-cat, β-catenin; APC, adenomatous polyposis coli; GSK-3β, glycogen synthase kinase 3β; Min, multiple intestinal neoplasia; YAMC, young adult mouse colon; IMCE, Immorto-Min colonic epithelium; EMSA, electrophoretic mobility shift assay; Tcf, T cell factor; Lef, lymphoid-enhancing factor; FAP, familial adenomatous polyposis; FBS, fetal bovine serum; TBS, Tris-buffered saline; ARF, alternative reading frame; INK, inhibitors of CDK4.
We thank Dr. Paul Polakis (Genentech, San Francisco, CA) for the myc-tagged ΔN89β-catenin construct and Dr. Robert Whitehead (Vanderbilt University, Nashville, TN) for the IMCE and YAMC cells.