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[Cancer Research 61, 7101-7109, October 1, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Alterations of ß- and {gamma}-Catenin in N-Butyl-N-(-4-hydroxybutyl)nitrosamine-induced Murine Bladder Cancer1

Hiroaki Shiina, Mikio Igawa, Shinji Urakami, Kazushi Shigeno, Tatsuaki Yoneda, Masaharu Terashima, Masao Deguchi, Leopoldo Ribeiro-Filho and Rajvir Dahiya2

Departments of Urology and Biochemistry, Shimane Medical University, Izumo, 693-0085 Japan [H. S., M. I., S. U., K. S., T. Y., M. T.], and Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, California 94121 [M. D., L. R-F., R. D.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abnormal degradation of ß-catenin caused by alteration of the glycogen synthase kinase-3ß (GSK-3ß) consensus motif is an important step for carcinogenesis. We hypothesize that ß- and {gamma}-catenin may play an important role in the pathogenesis of bladder cancer. We tested this hypothesis through analysis of ß- and {gamma}-catenin in both murine and human bladder cancers. A murine bladder cancer model was prepared by use of N-butyl-N-(-4-hydroxybutyl)nitrosamine (BBN) in 6-week-old male B6D2F1 mice. After 4, 8, 12, 16, 20, 24, and 28 weeks of BBN treatment, bladder specimens were harvested and analyzed for both protein and gene expression for ß- and {gamma}-catenin. Mutational analysis of the NH2-terminal regulatory domains of ß- and {gamma}-catenin was performed in each specimen by PCR-single-strand conformational polymorphism (SSCP) analysis. Mutations were further confirmed by direct DNA sequencing with a dye terminator method. Human bladder cancer specimens with normal tissues, dysplasia, carcinoma in situ, and carcinoma of grades, 1, 2, and 3 were also analyzed for ß- and {gamma}-catenin expression. ß- and {gamma}-catenin were analyzed for mutations by SSCP and direct DNA sequencing. Intracellular accumulation of ß- and {gamma}-catenin was observed in 6 of 20 invasive carcinoma specimens. There was no intracellular accumulation of ß- and {gamma}-catenin in mucosal dysplasia, papillary or nodular dysplasia, and carcinoma in situ specimens. On an SSCP analysis for ß-catenin, abnormal bandshifts were detected in two invasive carcinomas with intracellular ß-catenin accumulation. Further sequencing revealed two mutations [AGT(S) to ATT(I) and TCT(S) to CCT(P)] within the consensus motif for GSK-3ß phosphorylation. On the other hand, SSCP analysis for {gamma}-catenin followed by sequencing revealed three mutations in two invasive carcinomas with intracellular accumulation of {gamma}-catenin. These three alterations affected the 3' downstream region outside the GSK-3ß phosphorylation site [ACC(T) to GCC(A), CTC(L) to ATC(I), and CTC(L) to ATG(M)]. In human bladder cancer, ß- and {gamma}-catenin expression was significantly weaker than in normal bladder. On SSCP analysis one abnormal bandshift was observed in high-grade human bladder cancer with intracellular ß-catenin accumulation. DNA sequencing revealed mutation TCT(S) to TGT(C). In summary, alterations in ß- and {gamma}-catenin are late events favoring tumor progression in mouse BBN-induced bladder cancer. Changes affecting the GSK-3ß phosphorylation site appear to be associated with activation of ß-catenin, but not with activation of {gamma}-catenin. In human blabber cancer, ß- and {gamma}-catenin expression is similar to the expression in the mouse model. The present study demonstrates that ß- and {gamma}-catenin may play an important role in bladder cancer progression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanisms of metastasis and/or invasion of tumor cells involve multiple and complicated steps. ß- and {gamma}-catenin have been known to serve a pivotal role in this process (1, 2, 3, 4) . Studies have shown that loss of ß- and {gamma}-catenin function is associated with tumor metastasis (5, 6, 7) . Recent reports have suggested that ß-catenin functions as a regulator of signal transduction in addition to its classical role as a regulator of cell adhesion system (8) . Association of altered ß-catenin with tumor development has been emphasized in colorectal cancer (9 , 10) and liver tumors (11) . In colorectal cancer, intracellular accumulation of ß-catenin is caused either by mutational changes in the APC3 tumor suppressor gene or by mutation of ß-catenin gene itself (12 , 13) . On the other hand, in liver cancer, alteration of the ß-catenin gene itself predominantly occurs and results in tumor development (14) . Thus, in both tumors, either increased free ß-catenin caused by mutation of the APC gene or followed by alteration of the consensus motif for GSK-3ß phosphorylation as a result of the ß-catenin mutation itself is related to tumor progression.

{gamma}-Catenin, another cadherin-associated molecule, is also associated with the APC gene (1) and exhibits signaling activity similar to that of ß-catenin in Xenopus (15) . In human tumors, in contrast to ß-catenin, whether {gamma}-catenin is associated with signal transduction remains to be elucidated. Although there is a strong homology between ß- and {gamma}-catenin (1) , the functional role as a regulator of cell attachment appears to be somewhat different between the two. In bladder cancer, despite the high level of homology preserved between ß- and {gamma}-catenin, prognostic relevance has been noted only for {gamma}-catenin, but not for ß-catenin (16) , which might reflect potential differences in the functional roles or activated pathways between ß- and {gamma}-catenin.

In BBN-induced bladder cancer, tumor development progresses from mucosal dysplasia, PN dysplasia, and CIS, to invasive carcinoma when BBN is administrated to the mouse, representing a suitable model for invasive bladder cancer (17) . There has been no published report of the expression and mutation of ß- and {gamma}-catenin in the BBN-induced murine bladder cancer model. We hypothesize that alterations in ß- and {gamma}-catenin may play an important role in malignant transformation of the bladder. Using a BBN-induced murine bladder cancer model, we designed the present study to investigate whether alterations of ß- and {gamma}-catenin affect tumor initiation and/or tumor progression. We also compared the murine bladder cancer model with human bladder cancer through analysis of ß- and {gamma}-catenin expression and mutational analysis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
BBN-induced Murine Bladder Cancer Model.
Male B6D2F1 mice (obtained from Charles River, Kanagawa, Japan) were used in this study. The BBN-induced mouse bladder cancer model was prepared according to the method described by McCormick et al. (18) with some modifications. Briefly, 6-week-old male B6D2F1 mice (n = 84) were divided into two groups: the BBN treatment group (n = 70) and the control group (n = 14), which did not receive BBN treatment. In the BBN-treated mice, tap water containing 0.05% BBN was supplied ad libitum for 12 weeks, and thereafter tap water without BBN was given similarly. In control mice, water without BBN was given throughout the experiment. In both groups, the mice were sacrificed at 4, 8, 12, 16, 20, 24, and 28 weeks after the cessation of BBN treatment. Bladder specimens were harvested and analyzed for protein (immunohistochemistry) and DNA (mutation and sequencing).

Human Bladder Cancer Tissues.
Specimens were obtained from patients with primary TCC of the bladder who underwent total cystectomy. Each cystectomy specimen was opened along the midline of the anterior bladder wall and then fixed in 10% buffered formalin (pH 7.0) for at least 4 h but no more than 12 h after being pinned on a corkboard. After fixation, the entire bladder was cut into 2 x 0.5-cm strips. Each strip was embedded in paraffin wax and cut into 5-µm-thick consecutive sections; each section was used for immunostaining of ß- and {gamma}-catenin as well as for H&E staining for histological evaluation. Five different bladder specimens were used, and from each entire bladder specimen, at least five representative regions of normal mucosa, dysplasia, CIS, and TCC were selected based on pathological findings. All of these specimens were used for protein and DNA experiments. All of the tumor specimens were staged and graded histologically according to the TNM classification of malignant tumors of the Union International Contre Cancer (19) .

Immunohistochemistry.
Immunostaining of ß- and {gamma}-catenin was performed on 5-µm-thick consecutive sections obtained from paraffin-embedded materials according to the polymer immunocomplex method (20) . Sections were dried at room temperature, deparaffinized, rehydrated, and then treated with 2% hydrogen peroxide in methanol for 5 min at 37°C. Antigen retrieval was done by pepsin treatment (Biomed Corp., Foster City, CA) for 10 min at 40°C. After blocking with 3% normal goat serum, the sections were incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 37°C. Primary mouse monoclonal antibodies raised against ß-catenin (1:100 dilution) and {gamma}-catenin (1:500 dilution) were diluted with 5% mouse serum in PBS. Sections were washed with automation buffer (Biomed Corp) and then incubated with secondary antibody for 20 min at 37°C. Immunostaining was performed using the avidin-biotin-peroxidase method (Lab Vision, Fremont, CA) with diaminobenzidine as the chromogen and followed by counterstaining with hematoxylin. Normal mouse serum was used as a negative control. APC immunostaining was also performed using rabbit polyclonal antibody raised against a peptide mapping at the COOH terminus of human APC (Santa Cruz Biotechnology).

DNA Purification and PCR.
Genomic DNA from mouse bladder was purified and used as a template for PCR. The sequence information of mouse {gamma}-catenin, especially at the NH2 terminus, was not available. The sequence homology between human and rat {gamma}-catenin was first analyzed, especially in view of the known region encompassing the consensus motif for GSK-3ß phosphorylation in the mouse ß-catenin gene (the corresponding amino acid sequence is SYLDSGIHSGATTTA PSLSG). Considering the potential similarity between mouse and rat DNA sequence, the primer sets amplifying the regions of the interest were then determined. PCR primers for mouse ß- and {gamma}-catenin, encompassing the GSK-3ß phosphorylation motif at the NH2-terminal region, were as follows: forward primer for mouse ß-catenin, 5'-gctgacctgatggagttgg-3'; reverse primer, 5'-cttgcycttgcgtgaagg-3'; forward primer for {gamma}-catenin, 5'-cacgatggaggtgatgaacc-3'; reverse primer, 5'-gtcatgtgtgagttcttttga-3'.

Genomic DNA from human bladder tissues was purified and used as a template for PCR. Exon 3 of the ß-catenin gene, which encompasses the sequence for GSK-3ß phosphorylation (primers: forward, 5'-atttgatggagttggacatggc-3'; reverse, 5'-ccagctacttgttcttgagtgaagg-3') and a genomic fragment encoding amino acids 1–57 of {gamma}-catenin and encompassing the NH2-terminal regulatory region (primers: forward, 5'-ctcagtagccacgatggaggtg-3'; reverse, 5'-ttcttgagcgtgtactggcg-3') were amplified by PCR as described previously (13 , 21) . Each genomic DNA sample (2 µl) was diluted into 20 µl of solution containing 200 mM deoxynucleotide triphosphates, 500 nM each primer, 0.5 units of Taq polymerase, and PCR reaction buffer. For mutational analysis by SSCP, samples were amplified through 35 cycles on a Thermal Cycler (MJ Research) with denaturation at 94°C for 30 s; annealing for mouse and human ß-catenin at 60°C for 30 s, for mouse {gamma}-catenin at 58°C for 30 s, and for human {gamma}-catenin at 57°C for 30 s; and extension at 72°C for 45 s. In each set of PCR reactions, a tube without template DNA served as a negative control. Amplified PCR products were separated on 2% agarose gels containing 2 mg/ml ethidium bromide in Tris-borate-EDTA buffer and examined using a UV transilluminator.

SSCP Analysis and Direct Sequencing.
For mutational analysis, PCR products were used for SSCP and direct DNA sequencing. Briefly, equal volumes of each PCR product and SSCP buffer consisting of 850 µl of formamide, 50 µl of 10 mM EDTA (pH 8.0), 50 µl of 1.5% Ficoll in formamide, 50 µl of 0.05% bromphenol blue, and 0.05% xylene cyanol were mixed. After being denatured at 95°C for 5 min, the mixture was immediately placed on ice. A half-volume of the mixture was then loaded onto a 12% polyacrylamide gel. Electrophoresis was at 18°C and 25°C for 5 h at a constant 55W in Tris-glycine buffer. The gels were stained, and the samples with abnormal bandshifts were incubated with distilled water for 20 min at 95°C. The second PCR, which used the supernatant of this gel solution as a template, was carried out under the same conditions except for the number of PCR cycles (25 cycles). Excess primers and deoxynucleotide triphosphates in PCR products were removed by a Microcon tube (Millipore). Purified PCR products were used as templates for direct sequencing. DNA sequencing was carried out using a dye terminator method according to the manufacturer’s instructions (Applied BioSystem).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BBN-induced Murine Bladder Cancer Model.
The BBN-induced murine bladder cancer model was histologically defined as follows: (a) normal urothelial mucosa, characterized by epithelium of less than three layers without any anaplasia (Fig. 1a)Citation ; (b) mucosal dysplasia, characterized by epithelium of more than four layers with moderate or severe anaplasia demonstrating diffuse proliferation (Fig. 1b)Citation ; (c) PN dysplasia, characterized by moderate or severe anaplastic epithelial lesion of localized cellular proliferation resulting in nodular or papillary forms involving enfolding of capillaries and connective tissues (Fig. 1c)Citation ; (c) CIS, characterized by severe epithelial cellular atypia with mitosis and loss of polarity not including nodular or papillary proliferation; and (d) invasive carcinoma infiltrating the submucosa or muscle layer with transitional or partly squamous cell features (Fig. 1d)Citation .



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  		Fig. 1. Histology of BBN-induced murine bladder cancer. a, normal urothelial mucosa (x200). b, representative area of mucosal dysplasia in a mouse 12 weeks after BBN treatment (x400). c, representative areas of PN dysplasia in a mouse 20 weeks after BBN treatment (x200). d, representative area of invasive carcinoma in a mouse 24 weeks after BBN treatment (x200).

 
The intracellular accumulation of ß-and {gamma}-catenin in dysplasia, PN dysplasia, CIS, and invasive carcinoma of BBN-induced murine bladder cancer is summarized in Table 1Citation . Mucosal dysplasia, PN dysplasia, CIS, and invasive carcinomas were found in 43, 40, 19, and 15 mice, respectively, by the end of 28 weeks after cessation of BBN administration. The first mucosal dysplasia and PN dysplasia developed in the mice at 8 weeks after cessation of BBN treatment. On the other hand, the development of CIS and invasive carcinoma were later events, i.e., the first CIS and invasive carcinoma were observed in the mice at 16 and 20 weeks after cessation of BBN treatment, respectively.


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  		Table 1 Intracellular accumulation of ß- and {gamma}-catenin in BBN-induced murine bladder cancer

 
Intracellular accumulation of ß-catenin was detected in the six specimens of those mice with invasive carcinomas developed >20 weeks after cessation of BBN treatment (two each at 20, 24 and 28 weeks). In addition, in these six carcinomas with intracellular ß-catenin accumulation, four carcinomas preserved cytoplasmic APC immunoreactivity, whereas the remaining two showed either weak or lost APC immunoreactivity (Fig. 2Citation and Table 2Citation ). Similarly, in the regions of invasive carcinomas, intracellular accumulation of {gamma}-catenin was observed in one, three, and two specimens of those mice at 20, 24, and 28 weeks after cessation of BBN treatment, respectively. Two of six carcinomas with intracellular {gamma}-catenin accumulation showed weak or lost APC immunoreactivity, whereas APC immunoreactivity was preserved in the remaining four carcinomas (Table 2)Citation . Interestingly, two of six invasive carcinomas showed intracellular accumulation of both ß- and {gamma}-catenin (Table 2)Citation . As a whole, the distribution of tumor cells with intracellular accumulation of ß- or {gamma}-catenin immunoreactivity was rather heterogeneous (Fig. 3, c and dCitation , and Fig. 4dCitation ). In particular, the tumor cells with nuclear localization of ß- or {gamma}-catenin were focally distributed in the invasive carcinoma tissues (Fig. 3, e and fCitation , and Fig. 4, e and fCitation ).



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  		Fig. 2. Immunostaining of APC in BBN-induced murine bladder cancer (a–d) and human bladder cancer (e–h). a, PN dysplasia in a mouse 20 weeks after BBN treatment (x400) shows strong intracellular localization of APC. b, invasive carcinoma 24 weeks after BBN treatment (x400) shows reduced APC. c, invasive carcinoma 28 weeks after BBN treatment (x200) shows lack of APC in majority of cells. d, invasive carcinoma 28 weeks after BBN treatment (x400) shows no APC immunoreactivity in tumor cells, whereas interstitial tissues show weak cross-immunoreactivity. e, normal urothelial mucosa in human bladder (x200) shows strong APC expression. f, TCC G1 in case 1 (x200) shows strong APC expression, where the localization is in the cytoplasm rather than in the nuclei. g, TCC G2 in case 3 (x400) shows strong APC immunoreactivity, and the localization is in the nuclei rather than in the cytoplasm. h, TCC G3 in case 3 (x400) shows strong intracellular localization of APC in the invasive front.

 

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  		Table 2 Correlation of intracellular accumulation of ß- and {gamma}-catenin with mutational status and APC immunostaining in murine bladder cancer model

 


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  		Fig. 3. Immunostaining of ß-catenin in BBN-induced murine bladder cancer. a, dysplasia in a mouse 12 weeks after BBN treatment (x400). Strong membranous staining of ß-catenin is shown. b, PN dysplasia in a mouse 16 weeks after BBN treatment (x400). Membranous staining of ß-catenin is shown. c, invasive carcinoma in a mouse 24 weeks after BBN treatment (x200). Heterogeneous expression of ß-catenin is shown. Membranous staining of ß-catenin is reduced, whereas weak cytoplasmic immunoreactivity is seen in the majority of carcinoma cells. Some carcinoma cells show a strong nuclear staining (arrow). d, invasive carcinoma in a mouse 28 weeks after BBN treatment (x200). The majority of carcinoma cells show cytoplasmic immunoreactivity for ß-catenin. A small subset of carcinoma cells show a strong nuclear immunoreactivity of ß-catenin (arrow). Note that membranous staining of ß-catenin is weak and/or lost. e, invasive carcinoma in a mouse 24 weeks after BBN treatment (x400) shows reduced membranous staining of ß-catenin and strong nuclear accumulation of ß-catenin (arrow). f, invasive carcinoma in a mouse 28 weeks after BBN treatment (x400) shows strong nuclear and cytoplasmic staining (arrow).

 


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  		Fig. 4. Immunostaining of {gamma}-catenin in BBN-induced murine bladder cancer. a, normal urothelial mucosa (x200) shows strong membranous staining of {gamma}-catenin. b, dysplasia in a mouse 12 weeks after BBN treatment (x400) shows strong membranous staining of {gamma}-catenin. c, PN dysplasia in a mouse 20 weeks after BBN treatment (x200). Dysplastic cells with strong membranous staining of {gamma}-catenin are homogeneously distributed. d, invasive carcinoma in a mouse 24 weeks after BBN treatment (x200). Membranous staining of {gamma}-catenin is lost. The sporadic distribution of cancer cells with strong intracellular accumulation of {gamma}-catenin is shown (arrow). e, invasive carcinoma 24 weeks after BBN treatment (x400) shows strong intracellular localization of {gamma}-catenin (arrow). f, invasive carcinoma 28 weeks after BBN treatment (x400) shows strong cytoplasmic staining of {gamma}-catenin (arrow).

 
Histological Findings and Immunostaining of Human Bladder Cancer.
The histological findings and immunostaining of ß- and {gamma}-catenin are summarized in Table 3Citation .


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  		Table 3 Correlation of clinical history, pathological grades, expression of ß- and {gamma}-catenin, APC expression, and mutations of ß- and {gamma}-catenin in human bladder cancer

 
In case 1 (71-year-old male; pT1G1-2 TCC), extensive distribution of TCC and dysplasia was noted. Membranous staining of ß- and {gamma}-catenin and APC immunoreactivity were rather well preserved even in the dysplastic, CIS, and TCC regions. There was no cytoplasmic or nuclear staining of ß- and {gamma}-catenin in this case.

In case 2 (71-year-old female; pT1G2-3 TCC), many areas with mucosal ablation were observed, and dysplasia and CIS were extensive. The relationship of mucosal changes with ß- and/or {gamma}-catenin and APC immunoreactivity was almost the same as observed in case 1. No apparent difference was observed in ß- or {gamma}-catenin immunostaining between the G2 and G3 TCC regions.

In case 3 (69-year-old male; pT3bG2-3 TCC), the normal mucosa was widely distributed in the vesical cavity. Neoplastic changes were confined to the trigone and bladder neck, and involvement of lymphatic vessels was noted at the invasive front. Membranous staining of ß- and {gamma}-catenin and APC immunoreactivity were rather well preserved even in the dysplastic, CIS, and TCC regions (Fig. 2, e and fCitation , and Fig. 5, aCitation ). Interestingly, cytoplasmic and/or nuclear staining of ß- and {gamma}-catenin was noted in the tumor cells at the TCC G3 invasive front (Fig. 5, b, c, e, and f)Citation . However, this intracellular localization of ß- and {gamma}-catenin was not found in regions of normal transitional epithelium, dysplasia, CIS, or TCC G2. Even at the TCC G3 invasive front, APC immunoreactivity was well preserved (Fig. 2, g and h)Citation .



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  		Fig. 5. Immunostaining of ß- and {gamma}-catenin in human bladder cancer. a, in the regions of TCC G2, relatively well-preserved membranous ß-catenin staining is seen (x400). b and c, invasive carcinoma front (TCC G3) shows strong intracellular accumulation of ß-catenin (b, x200; c, x400). d, in the regions of TCC G2, membranous staining is observed with partial loss in some tumor cells. No intracellular accumulation was found (x400). e and f, at the invasive front, some tumor cells show intracellular accumulation of {gamma}-catenin (e, x200; f, x400).

 
In case 4 (67-year-old female; pT3bG3 TCC), CIS, dysplasia, and TCC G3 were widely distributed. Membranous staining of ß-catenin was rather strong even in the regions of CIS, dysplasia, and TCC G3. Membranous staining of {gamma}-catenin was strong in normal transitional epithelium, but was weak or lost in the dysplastic, CIS, and TCC regions. APC immunoreactivity was weak in the TCC G3 region. Neither cytoplasmic nor nuclear staining of ß- and {gamma}-catenin was found.

In case 5 (50-year-old male; pT1G2–3 TCC), the main tumor was located in the posterior wall. Membranous staining of ß- and {gamma}-catenin was rather well preserved even in dysplastic, CIS, and TCC regions compared with normal transitional epithelium. Neither cytoplasmic nor nuclear staining of ß- and {gamma}-catenin was observed in normal epithelium or in dysplastic, CIS, or TCC regions. The staining intensity of ß- and {gamma}-catenin did not differ between G2 and G3 TCCs. APC immunoreactivity was not significantly different among these regions.

SSCP Analysis of ß- and {gamma}-Catenin in Murine Bladder Cancer.
Fifty-nine bladder specimens were subjected to PCR-SSCP analysis. In SSCP analysis for ß-catenin, there were no abnormal bands detectable in the regions of mucosal dysplasia, PN dysplasia, and CIS. In two of six invasive carcinomas, bandshift was observed for ß-catenin (Fig. 6a)Citation . ß-Catenin immunoreactivity of these two specimens showed strong intracellular accumulation (Fig. 2, e and f)Citation . The remaining four of six invasive carcinomas with intracellular ß-catenin accumulation did not exhibit abnormal bands on SSCP (Table 2)Citation . Direct sequencing revealed two different single-base substitutions resulting in amino acid changes (each serine was substituted for isoleucine or proline in these specimens; Fig. 7Citation ). As shown in Fig. 8Citation , these two amino acid changes were in the region of consensus motif for GSK-3ß phosphorylation.



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  		Fig. 6. Results of PCR-SSCP analysis for BBN-induced murine bladder cancer. a, ß-catenin SSCP. There were no aberrant bandshifts in the regions of mucosal dysplasia (D), PN dysplasia (PN-D), and CIS, compared with the band in the control genomic DNA (C). In invasive carcinomas an additional aberrant band was observed in two of six specimens. b, {gamma}-catenin SSCP. There was no bandshift in mucosal dysplasia (D), PN dysplasia (PN-D), and CIS, compared with the band in the control genomic DNA (C). In invasive carcinoma, two of six specimens showed bandshifts.

 


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  		Fig. 7. Direct sequencing of abnormal bands on a SSCP analysis for ß-catenin. a, invasive carcinoma 24 weeks after BBN treatment with intracellular accumulation of ß-catenin (corresponding to Fig. 5aCitation ) shows a single-base substitution (G to T) that causes an amino acid change from serine (left) to isoleucine (right). b, invasive carcinoma 28 weeks after BBN treatment with intracellular accumulation of ß-catenin (corresponding to Fig. 5bCitation ) shows a single-base substitution (T to C) that causes an amino acid change from serine (left) to proline (right).

 


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  		Fig. 8. Putative consensus motif for the GSK-3ß phosphorylation site (overlined letters) and neighboring amino acid sequence in mouse ß- and {gamma}-catenin. Shown are alterations of the amino acids, caused by a ß- or {gamma}-catenin mutation, and phosphorylation sites for PKC.

 
For {gamma}-catenin, there was no mutation in the specimens obtained from the regions of mucosal dysplasia, PN dysplasia, and CIS. Interestingly, one of six invasive carcinomas showed bandshift (Fig. 6b)Citation . In one invasive carcinoma at 24 weeks after cessation of BBN treatment, {gamma}-catenin immunoreactivity was strongly accumulated in the cytoplasm (Fig. 4f)Citation . As shown in Fig. 9aCitation , direct sequencing of this invasive carcinoma showed that C and A were substituted for T and G, respectively, within the consecutive two codons, resulting in one amino acid change from threonine to alanine. On the other hand, in the remaining invasive carcinoma at 28 weeks after cessation of BBN treatment, {gamma}-catenin immunoreactivity was observed in the nuclei rather than in the cytoplasm (Fig. 4e)Citation . Direct sequencing of this invasive carcinoma revealed that three C bases were substituted for A, A, and G, respectively, within the consecutive two codons, changing the amino acids from leucine to isoleucine and from leucine to methionine (Fig. 9b)Citation . As shown in Fig. 8Citation , these three amino acid changes were in the downstream regions outside the GSK-3ß phosphorylation site.



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  		Fig. 9. Direct sequencing of abnormal bands in SSCP analysis for {gamma}-catenin. a, invasive carcinoma 24 weeks after BBN treatment with intracellular accumulation of {gamma}-catenin (corresponding to Fig. 5cCitation ) shows two different base substitutions (C to T and A to G); the former is a silent mutation and the latter causes an amino acid change from threonine to alanine. b, invasive carcinoma 28 weeks after BBN treatment with intracellular accumulation of {gamma}-catenin (corresponding to Fig. 5dCitation ) shows C substitutions, at three different nucleotide positions, for A, A, and G, resulting in two amino acid changes, from leucine to isoleucine and from leucine to methionine.

 
SSCP Analysis of ß- and {gamma}-Catenin in Human Bladder Cancer.
One of five cases showed bandshift in exon 3 of the ß-catenin gene. This specimen was from the invasive front of a G3 tumor (Fig. 10a)Citation . Additional sequencing data of the abnormal band confirmed a TCT-to-TGT mutation at codon 37, changing a highly conserved serine to cysteine (Fig. 10b)Citation . As shown in Fig. 10Citation , this amino acid change was in the region inside the consensus motif for GSK-3ß phosphorylation. However, any mutational changes of the NH2-terminal regulatory region of {gamma}-catenin were detected by SSCP analysis in our materials (Fig. 10c)Citation . The relationship of the SSCP analysis with the immunostaining and histological findings is summarized in Table 3Citation .



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  		Fig. 10. Results of PCR-SSCP analysis and direct sequencing in human bladder cancer. a, detection of a ß-catenin mutation in exon 3 in case 3 by SSCP analysis. Lane WT contains the fragments with normal mobility. Lanes 1–5 contain DNA extracted from regions of normal transitional epithelium, dysplasia, CIS, G2 TCC, and G3 TCC at the invasive front, repsectively. An abnormally shifted band is shown in Lane 5 (arrow). b, sequence chromatogram of ß-catenin of G3 TCC at the invasive front in case 3 (Lane 5 in a) shows changes from TCT (serine) to TGT (cysteine) at codon 37 (arrow). c, SSCP analysis of the NH2 terminal regulatory region of {gamma}-catenin. No aberrant bandshifts were detected. Lane WT contains the fragments with normal mobility. Lanes 1–5 contain DNA extracted from cancer regions of cases 1, 2, 3, 4, and 5, respectively.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ß-Catenin functions as a regulator for the cell-cell adhesion system and signal transduction (1 , 12 , 13) . Regulation of nonmembrane-bound free pools of ß-catenin by the Wnt signaling pathway has been extensively investigated (22) . Furthermore, intracellular ß-catenin is degraded by phosphorylation through the ubiquitin proteasome system (23) . Tumor initiation and/or progression through intracellular ß-catenin accumulation caused by alteration of the coding region for the GSK-3ß phosphorylation site of the ß-catenin gene has been considered as an alternative to APC gene mutations in some solid tumors, including colorectal and liver cancer (12 , 13) . Thus, this posttranslational event based on alteration of the consensus motif for the GSK-3ß phosphorylation site is one of the mechanisms related to tumor progression (12, 13, 14) . However, for {gamma}-catenin, a molecule with close similarity to ß-catenin with respect to sequence homology and its function as mediator for cell-cell attachment (1, 2, 3, 4) , whether {gamma}-catenin exerts an influence on tumor initiation and/or progression remains to be studied. Using the BBN-induced mouse bladder cancer model, we investigated (a) whether an alteration in ß-catenin is involved during mouse bladder carcinogenesis and (b) whether {gamma}-catenin is associated with bladder cancer initiation and/or progression and, if it is involved, whether the process is the same as that involved in ß-catenin alteration. In addition, we compared the results obtained from the BBN-induced murine bladder cancer model with human bladder cancer.

The steps for carcinogenesis in the BBN-induced murine bladder cancer model have been well studied (18) . Bladder mucosa after BBN treatment is subjected to several pathological changes, from normal urothelium through mucosal dysplasia, PN dysplasia, CIS, and finally to invasive carcinoma. These lesions mimic those observed in serious invasive TCC in humans. Genetically, the frequency of p53 mutation in BBN-induced bladder carcinoma (24) is similar to that observed in human invasive bladder carcinomas (25) . In addition, inactivation of p16 may be an early event in both human and BBN-induced mouse bladder carcinogenesis (24) .

In the present study, we found that the reduced membranous staining of both ß- and {gamma}-catenin was well correlated with the pathological changes of bladder mucosa; in normal mucosa membranous staining was well preserved, whereas it was reduced or lost in invasive carcinomas (Figs. 3Citation and 4Citation ). This finding reflected the function of ß- and {gamma}-catenin as a regulator of cell-cell attachment and was in accordance with the previous reports (7 , 16) . On the contrary, intracellular accumulation of both ß- and {gamma}-catenin was found only in the regions of invasive carcinomas, whereas it was not found in the regions of mucosal dysplasia, PD dysplasia, and CIS. Thus, only invasive phenotypes of BBN-induced bladder cancer showed intracellular accumulation of ß- and {gamma}-catenin. These findings appear to indicate that intracellular accumulation of ß- and {gamma}-catenin is associated with tumor progression but rarely with tumor initiation. On the other hand, changes of membranous staining of ß- and {gamma}-catenin in human bladder cancer (Table 3)Citation were very similar to those observed in the BBN-induced murine bladder cancer model. In addition, intracellular accumulation of ß- and {gamma}-catenin was restricted to the region of TCC G3, especially at the invasive front. Thus, intracellular localization of ß- and {gamma}-catenin is also related to tumor progression, but not to tumor initiation in human bladder cancer.

We further investigated how intracellular accumulation of ß- and {gamma}-catenin is associated with tumor progression in mouse bladder cancer. It has been reported that one of the mechanisms for intracellular accumulation of ß-catenin is mutational alteration of the GSK-3ß phosphorylation site in the ß-catenin gene (3 , 26) . Although ß-catenin is regulated through phosphorylation by GSK-3ß, a mutation in ß-catenin encompassing the region containing the GSK-3ß phosphorylation site results in the intracellular accumulation of stabilized ß-catenin (26) . In our study, 6 of 15 invasive carcinomas showed intracellular accumulation of ß-catenin immunoreactivity. Of these six invasive carcinomas, only two mutational alterations in ß-catenin were detectable. Both affected the consensus motif for the GSK-3ß phosphorylation site, and both involved serine residues. Therefore, in these two invasive carcinomas, poorly phosphorylated ß-catenin was increased, translocated, and accumulated in the nucleus. This activated ß-catenin could enhance the downstream target genes for the Wnt/ß-catenin signal, increasing cell proliferation (27) . This phenomenon has typically been observed in colon cancer (12 , 13) .

Recently, c-myc and cyclin D1, the gene products of which are functionally associated with cellular proliferation in various solid tumors, have been demonstrated as target genes for the Wnt signal (28 , 29) . In the remaining four invasive carcinomas, which lacked ß-catenin mutations but exhibited intracellular accumulation, rather heterogeneous and/or focal staining patterns of ß-catenin accumulation were observed. ß-Catenin might reflect transient activation of the Wnt signaling pathway. In fact, frequent intracellular localization of ß-catenin in the absence of a ß-catenin mutation has been reported in malignant melanoma (30) . An alternative possibility is that mutational alterations in the elements related to the Wnt signaling pathways (GSK-3ß, APC, and axin) could involve intracellular accumulation of ß-catenin. Phosphorylation by GSK-3ß involves various key growth-regulatory proteins related to insulin as well as growth factor signaling (31 , 32) . The inactivation of GSK-3ß could be a fatal event to the cell. In fact, mutations resulting in GSK-3ß inactivation have not been reported. Thus, association of APC immunostaining with intracellular ß-catenin accumulation was studied. As shown in Table 2Citation , two invasive carcinomas showed weak or lost APC immunoreactivity, but they harbored no ß-catenin mutations. In these carcinomas, the functional loss of APC protein might be attributed to the intracellular accumulation of ß-catenin. On the other hand, in two invasive carcinomas, intracellular ß-catenin accumulation was observed despite the absence of a ß-catenin mutation and preserved APC immunoreactivity. Recently, it has been reported that an inactivating mutation of axin alters ß-catenin regulation and that this event is another mechanism for the accumulation of free ß-catenin in hepatocellular carcinoma (33) . In these two carcinomas, it might be possible that alteration of axin might affect ß-catenin accumulation.

As for {gamma}-catenin, two of six invasive carcinomas with intracellular accumulation harbored {gamma}-catenin mutations (Table 2)Citation . As shown in Fig. 8Citation , however, these mutations affected the 3' downstream region outside the putative consensus motif for GSK-3ß phosphorylation. Considering that cytoplasmic dominant accumulation and mutations outside the GSK-3ß phosphorylation site were found only in invasive carcinomas, it is possible that it might occur through an independent pathway of GSK-3ß phosphorylation. The regions subject to three mutational changes of {gamma}-catenin were at two different sites for PKC phosphorylation (Fig. 8)Citation . A recent investigation has suggested that PKC activation has a strong impact on maximum accumulation of ß-catenin and transcriptional activation (34) . In addition, induction of {gamma}-catenin is considered to be dependent on some PKC isoforms (35) . It is possible that conformational and/or structural changes of {gamma}-catenin caused by mutations have a positive influence on the recognition by PKC. In the remaining four invasive carcinomas with intracellular accumulation, the possible mechanisms of accumulation appeared to be similar to those for ß-catenin accumulation, as mentioned above. It has been reported that axin promotes GSK-3ß-dependent phosphorylation of {gamma}-catenin and regulates the stability of {gamma}-catenin (36) . On the other hand, the NH2 terminus of the region amplified with our {gamma}-catenin primers demonstrated an alignment similar in part to the WW-domain signature (37) . Recently, it has been reported that some of the WW-domain proteins appear to be signaling proteins (38) and that an amino acid sequence with a similarity to the WW domain is present in some signal transaction proteins (39) . In the present study, ß- and {gamma}-catenin immunostaining and mutation analysis suggested that alteration of ß- and {gamma}-catenin might be a later event and affects a small subset of tumor cells during mouse bladder carcinogenesis induced by BBN. The detachment of tumor cells occurs at an early phase after BBN administration, whereas the tumor cells that escape from the cell attachment system acquire increased cellular proliferation through the activated Wnt signaling pathway at a later event.

In human bladder cancer, the mutation was a missense mutation of serine to cysteine at codon 37, which affected four highly conserved serine/threonine sites of phosphorylation by GSK-3ß that are crucial for ß-catenin. In addition, strong APC immunoreactivity was observed even at the invasive front (Fig. 2h)Citation . Therefore, the intracellular accumulation of ß-catenin in case 3 might correspond to an increased level of hypophosphorylated ß-catenin caused by a mutation, in turn leading to transduction of oncogenic signals and cancer progression. However, SSCP analysis of {gamma}-catenin revealed no mutational changes in the specimens irrespective of {gamma}-catenin nuclear accumulation. In addition to the possible alteration of axin, amplification or rearrangement of the {gamma}-catenin gene might be related to overexpression of {gamma}-catenin.

In summary, this is the first report that demonstrates the involvement of ß- and {gamma}-catenin in both BBN-induced murine bladder cancer and human bladder cancer. We further documented that alterations of ß- and {gamma}-catenin are associated with tumor progression rather than initiation. These results are important in understanding the pathogenesis of bladder cancer.


   FOOTNOTES
 
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 This study was supported by Grants RO1DK-55040 and RO1DK-51101 from NIH and a Veterans Affairs Merit review grant. Back

2 To whom requests for reprints should be addressed, at University of California San Francisco and VA Medical Center, 4150 Clement Street, San Francisco, CA 94121. Phone: (415) 750-6964; Fax: (415) 750-6639; E-mail: Urologylab{at}aol.com Back

3 The abbreviations used are: APC, adenomatous polyposis coli; GSK-3ß, glycogen synthase kinase-3ß; BBN, N-butyl-N-(-4-hydroxybutyl) nitrosamine; PN papillary or nodular; CIS, carcinoma in situ; TCC, transitional cell carcinoma; SSCP, single-strand conformational polymorphism; PKC, protein kinase C. Back

Received 4/28/01. Accepted 8/ 2/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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