Alterations of beta- and gamma-catenin in N-butyl-N-(-4-hydroxybutyl)nitrosamine-induced murine bladder cancer.
Abnormal degradation of beta-catenin caused by alteration of the glycogen synthase kinase-3beta (GSK-3beta) consensus motif is an important step for carcinogenesis. We hypothesize that beta- and gamma-catenin may play an important role in the pathogenesis of bladder cancer. We tested this hypothesis through analysis of beta- 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 beta- and gamma-catenin. Mutational analysis of the NH(2)-terminal regulatory domains of beta- 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 beta- and gamma-catenin expression. beta- and gamma-catenin were analyzed for mutations by SSCP and direct DNA sequencing. Intracellular accumulation of beta- and gamma-catenin was observed in 6 of 20 invasive carcinoma specimens. There was no intracellular accumulation of beta- and gamma-catenin in mucosal dysplasia, papillary or nodular dysplasia, and carcinoma in situ specimens. On an SSCP analysis for beta-catenin, abnormal bandshifts were detected in two invasive carcinomas with intracellular beta-catenin accumulation. Further sequencing revealed two mutations [AGT(S) to ATT(I) and TCT(S) to CCT(P)] within the consensus motif for GSK-3beta 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-3beta phosphorylation site [ACC(T) to GCC(A), CTC(L) to ATC(I), and CTC(L) to ATG(M)]. In human bladder cancer, beta- 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 beta-catenin accumulation. DNA sequencing revealed mutation TCT(S) to TGT(C). In summary, alterations in beta- and gamma-catenin are late events favoring tumor progression in mouse BBN-induced bladder cancer. Changes affecting the GSK-3beta phosphorylation site appear to be associated with activation of beta-catenin, but not with activation of gamma-catenin. In human blabber cancer, beta- and gamma-catenin expression is similar to the expression in the mouse model. The present study demonstrates that beta- and gamma-catenin may play an important role in bladder cancer progression.
Shiina
H
H
Department of Urology, Shimane Medical University, Izumo, 693-0085 Japan.
Igawa
M
M
Urakami
S
S
Shigeno
K
K
Yoneda
T
T
Terashima
M
M
Deguchi
M
M
Ribeiro-Filho
L
L
Dahiya
R
R
D000368:Aged; D000818:Animals; D002085:Butylhydroxybutylnitrosamine; D002273:Carcinogens; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D051180:Desmoplakins; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008875:Middle Aged; D018807:Polymorphism, Single-Stranded Conformational; D015534:Trans-Activators; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051176:beta Catenin; D051185:gamma Catenin
C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D002273:Carcinogens; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; C495292:JUP protein, human; C495293:Jup protein, mouse; D015534:Trans-Activators; D051176:beta Catenin; D051185:gamma Catenin; D002085:Butylhydroxybutylnitrosamine