Transformation and Morphological Changes of Murine L Cells by Transfection with a Mutated Form of β-Catenin¹ Free

In addition to functioning as an essential component of the cadherin-mediated cell-cell adhesion system (1), β-catenin plays an important role in cell growth by transmitting the wnt/wingless signal in embryonic cells (2). In differentiated cells, the cytoplasmic level of β-catenin is kept very low through degradation by the ubiquitin-proteasome pathway, whereby serine and threonine residues in exon 3 are phosphorylated by GSK3β³ and ubiquitinylated by binding to proteins such as APC, axin, and conductin (3, 4). In experiments with colorectal cancer and melanoma cell lines, dysfunction of APC induced stabilization of β-catenin, resulting in excess β-catenin bound to Tcf/Lef to activate transcription in the nucleus (5, 6). Furthermore, in cell lines having no APC mutations, mutations of the β-catenin gene that altered amino acid residues representing potential GSK3β phosphorylation sites could confer resistance to degradation and lead to intracellular accumulation of β-catenin. Activated cytoplasmic β-catenin, probably bound to Tcf/Lef, is thought to migrate into the nucleus and stimulate transcription of downstream genes in a constitutive manner (5, 6).

β-catenin mutations reported thus far in human tumors have been concentrated at specific GSK3β phosphorylation sites, namely Ser-33, Ser-37, Ser-45, Thr-45, and residues flanking codon 33 (codons 32 and 34). Interstitial deletions that remove these amino acids seem to be oncogenic in various types of tumors (7, 8, 9). All mutations of β-catenin detected in Japanese primary colorectal cancers in our previous study were interstitial deletions of all or part of exon 3 (10). Others have reported missense mutations at serine/threonine residues in exon 3 (5, 11).

On the basis of those results, we introduced a mutated form of β-catenin that lacked an entire exon 3 into L cells derived from normal s.c. tissue of a C3H/An mouse to elucidate the role of β-catenin activation in tumorigenesis. Biological alterations in response to the accumulation of mutant β-catenin were analyzed.

L cells established from mouse strain C3H/An were grown in DMEM (4500 mg/liter d-glucose; Nikken Biomedical Laboratory) containing 10% fetal bovine serum (FBS, Cansera, Canada) at 37°C in 5% CO₂.

Wild-type β-catenin cDNA containing the entire coding region and a mutated form of β-catenin cDNA lacking exon 3 were obtained by RT-PCR amplification with cloned Pfu polymerase (Stratagene, La Jolla, CA), using as templates RNAs derived from a normal human tissue and a colon cancer known to have this β-catenin mutation. The primer sequences used here were 5′-CCATACAACTGTTTTGAAAATCC-3′ and 5′-GTTCAGACAATACAGCTAAAGG-3′. The β-catenin cDNAs were cloned into pUHD10-3 vector under the regulation of a tetracycline-responsive promoter (Tet-off Gene Expression System). Briefly, expression of β-catenin is induced in the absence of tetracycline or doxycycline, but transcription is suppressed in cells that are cultured in medium containing either of these drugs.

Wild-type or mutant β-catenin cDNAs was each cotransfected by lipofection (Lipofectamine Plus Re-agent; Life Technologies, Inc.) with pUHD15-1Neo. Cells were selected in medium containing geneticin (800 μg/ml) as well as doxycycline (2 ng/ml). Geneticin-resistant colonies were cloned and expanded, yielding clonal lines of L-MT and L-N, which expressed mutant and normal β-catenin, respectively. As a control, L cells were also transfected with the empty vector pUHD10-3 (L-mock).

After treatment with trypsin-EDTA, viable cells were counted by the trypan blue exclusion method. Each time, three samples were counted, and the averages of those numbers were recorded.

Total RNAs were isolated from each cell line with Trizol Reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. A 3-μg aliquot of each total RNA was reverse-transcribed for single-strand cDNA using oligo(dT)₁₅ primer and Superscript II (Life Technologies, Inc.). Each single-strand cDNA was diluted for subsequent PCR amplification by monitoring GAPDH as a quantitative control. The PCR was carried out in a reaction volume of 20 μl for 4 min at 94°C for initial denaturing, followed by 25 (for GAPDH) or 28 (for β-catenin) cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, on the Gene Amp PCR system 9600 (Perkin-Elmer Corp.). The primer sequences used for amplification were 5′-CAACTACATGGTTTACATGTTC-3′ and 5′-GCCAGTGGACTCCACGAC-3′ for GAPDH and 5′-TGTTCCGAATGTCTGAGGAC-3′ and 5′-GTTCAGACAATAC AGCTAAAGG-3′ for β-catenin. PCR products were resolved in 0.8 or 2% agarose gels and visualized by staining with ethidium bromide.

Extraction of proteins from cell lines and Western blot analyses were performed as described previously (10). Briefly, 20 μg of total protein extracted from each of the three cell lines (L-MT, L-N and parental L cells) were electrophoresed, transferred to polyvinylidene difluoride membrane, and visualized with an anti-β-catenin monoclonal antibody (C199220; Transduction Laboratories, Lexington, KY). The cells were also cultured on glass slides, fixed with 100% methanol, and stained with anti-β-catenin monoclonal antibody. The second antibody was FITC-labeled rabbit antimouse IgG (F0261; DAKO).

Aliquots of 1 × 10⁵ or 5 × 10⁵ cells were expanded in six-well plates (35 mm per well). Cell numbers in each culture were counted every 24 h for up to 7 days. At least three independent experiments were performed.

We introduced expression vectors containing normal or mutant β-catenin cDNAs or a vector control into murine L cells that normally express little endogenous β-catenin and established stable cell lines in which the expression of β-catenin was under the control of the presence or absence of tetracycline. Five independent l-N cell lines and 12 independent l-MT cell lines were isolated. A semiquantitative RT-PCR analysis indicated that, in the absence of doxycycline [Dox(−)], β-catenin was more abundantly expressed in cells transfected with normal cDNA (l-N) and cells with mutated cDNA (l-MT) than in l-MT cells grown with doxycycline [Dox(+)] or parental L cells (Fig. 1,A). Fig. 1,B shows β-catenin expression examined by Western blotting. The levels of M_r 92,000 normal-sized β-catenin in the parental L cells (Lane 1) or l-N cells (Lane 2) were low. However, a high level of M_r 84,000 mutant β-catenin, which corresponds to a product lacking 76 amino acids encoded by exon 3, was observed in l-MT cells (Lane 3). The amount of mutant β-catenin was dramatically reduced when l-MT cells were cultured in the presence of doxycycline [Dox(+), Lane 4] or tetracycline [Tet(+), Lane 5]. Immunohistochemical staining using anti-β-catenin antibody confirmed accumulation of β-catenin in cytoplasm and nuclei of l-MT cells (Fig. 2,A), but no staining was observed in L cells, l-N cells, or l-mock cells (data not shown). As mutated β-catenin accumulated in l-MT cells, their appearance changed to a more cuboidal morphology from the fibroblast-like parental L cells, and the l-MT cells grew with more condensed cell-cell contact (Fig. 2 B). l-N cells and l-mock cells showed no morphological changes.

Growth curves of l-MT and parental L cells grown in medium containing 10% serum are shown in Fig. 3,A. When cells were seeded at low concentration (1.0 × 10⁵ cells per six-well dish), l-MT cells proliferated at a rate similar to that of parental L cells in the proliferation phase. However, when seeded densely (5.0 × 10⁵ cells per six-well dish), l-MT cells continued to proliferate after they reached confluence, whereas growth of l cells was arrested after day 3, and cell numbers gradually decreased. At day 5, L cells reached confluence in a monolayer, whereas l-MT cells became packed tightly, piling up to a multilayer (Fig. 3 B). Most L cells died by day 8, whereas l-MT cells kept growing and continued to increase in numbers.

To determine whether the difference in growth properties was caused by accumulation of mutated β-catenin, we compared the growth rates of l-MT cells grown with or without induced expression of mutated β-catenin; i.e., with or without 2 ng/ml doxycycline in the medium (Fig. 3 C). The growth rate of l-MT cells cultured with doxycycline, which suppresses expression of mutant β-catenin, was suppressed to a level similar to that of parental l cells; after confluence, cell numbers were obviously decreased after day 10, although l-MT cells continued to grow.

Growth curves of l-N and l-mock grown in medium containing 10% serum showed almost the same growth properties as parental L cells. Their growth rates were similar to that of parental L cells and l-MT cells. At day 6, l-N and l-mock reached confluence in a monolayer, and the numbers of both cells began to decrease at day 8. Their growth properties were not influenced by the presence of doxycycline in medium.

To test whether β-catenin is sufficient to support cell growth in place of serum components, we analyzed the growth of l-MT cells under serum-free conditions and in medium containing only 2% serum. Parental L cells and l-MT cells were seeded sparsely, and viable cells were counted (Fig. 4). Both L cells and l-MT cells failed to grow under serum-free conditions. However, in the medium containing 2% serum, l-MT grew more rapidly than L cells or l-MT Dox(+), although l-MT cells proliferated more slowly in 2% serum than in 10% serum. The growth of l-N and l-mock cells was as slow as that of parental L cells in the medium containing 2% serum, and neither of the cells was unable to grow under the serum-free condition.

Five l-N cells revealed almost the same characteristics, and 12 l-MT cells had some clonal varieties in the accumulation of mutant β-catenin. However, five different l-MT cells with a high level of β-catenin expression showed similar behavior and morphological changes mentioned above.

We have demonstrated here that a mutated form of β-catenin lacking an entire exon 3 accumulates in the cytoplasm and nucleus of transfected cells. Although the level of expression induced by this mRNA was slightly lower than that of normal β-catenin, mutant β-catenin protein was far more abundant in l-MT cells than the normal protein in l-N cells. l-N cells showed no accumulation of β-catenin, although RT-PCR experiments confirmed mRNA expression of the exogenous wild-type β-catenin in l-N cells. Hence, we assume that exogenous wild-type β-catenin was rapidly degraded.

The type of β-catenin mutation lacking exon 3 was considered to be a β-catenin-activating mutation like missense mutation occurred at serine or threonine residues in exon 3 reported in cell lines (10). These observations support the idea that such mutations result in accumulation of β-catenin in the cytoplasm at the posttranslational level, probably by allowing the protein to escape from its normal degradation pathway.

Although the transforming activity of N-truncated β-catenin was documented previously (12), the biological mechanism had not been clarified. We have demonstrated here that accumulation of mutated β-catenin in a cell alters cellular morphology and induces a multilayer growth pattern without any change in growth rate when the medium contains 10% serum. The mutation also decreases the dependence of l-MT cells on serum concentration. These phenomena are consistent with certain characteristics of some cancer cells; i.e., abnormalities of cell-to-cell adhesion or cell polarity and serum-independent cellular proliferation.

wnt-1 or wnt-2 gene products can induce morphological changes, increase saturation density and cell-cell adhesiveness, and speed serum-independent cellular proliferation in fibroblast-derived cell lines (13, 14, 15). Activation of β-catenin is known to be important in the wnt signal transduction pathway and to be directly linked to alterations in cellular growth. Here, we showed that accumulation of one type of mutant β-catenin was able to alter the morphological and proliferative character of murine L cells, and those changes may explain the ability of β-catenin to act as an oncogene on its own. We anticipate that the system we used to regulate expression of this gene will facilitate future attempts to characterize β-catenin mutations in terms of function and to identify downstream genes that are transcriptionally activated by the gene product. Results of such studies should increase our understanding of tumorigenesis in various types of tumors.