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.

CpG hypermethylation of the promoter region inactivates the estrogen receptor-beta gene in patients with prostate carcinoma.

BACKGROUND: The down-regulation of the estrogen receptor-beta (ERbeta) gene is associated with several malignancies, including prostate carcinoma. The purpose of the current study was to investigate the mechanisms of ERbeta inactivation through the analysis of CpG methylation of the promoter region of ERbeta gene. METHODS: ERbeta protein expression was examined by immunohistochemistry in 23 cases of human prostate carcinoma and 40 cases of benign prostatic hyperplasia (BPH). DNA was extracted from these tissues and processed for sodium bisulfite genomic sequencing. The percentage of methylation of CpG sites in the promoter region of ERbeta (-376 to -117), which contains 19 CpG sites, was determined from genomic sequencing data. The prostate carcinoma cell lines DU145 and ND1 were treated with the demethylating agent 5-AZAC and ERbeta mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: In BPH tissues, ERbeta protein expression was found mainly in epithelial cells. ERbeta protein expression was lacking in 83% of prostate carcinoma samples (19 of 23 samples) whereas all cases of BPH (40 of 40) demonstrated expression of ERbeta protein. The mechanism of inactivation of the ERbeta gene in prostate carcinoma was CpG methylation because the degree of methylation at all CpG sites within the promoter region between -376 and -117 was higher in prostate carcinoma samples compared with BPH tissues. Nine of 19 CpG sites within the promoter region of ERbeta displayed significant differences in methylation between prostate carcinoma and BPH samples. The prostate carcinoma cell lines appeared to lack ERbeta expression. However, 5-AZAC treatment restored ERbeta expression in those cell lines, suggesting that methylation inactivates the ERbeta gene in prostate carcinoma. CONCLUSIONS: The results of the current study demonstrate, for what we believe to be the first time, that the inactivation of the ERbeta gene in prostate carcinoma occurs through CpG methylation of the promoter region of this gene.

CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma.

CpG methylation in the promoter region has been shown to be important in the regulation of genes implicated in malignant transformation. The present study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression in renal cell carcinoma. To test this hypothesis, five kidney cancer cell lines and 34 microdissected renal cell carcinoma samples were analyzed for gene and protein expression by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. CpG methylation in the promoter regions of the E-cadherin gene was analyzed by the sodium bisulfite genome sequencing technique. Our results show that all normal renal tissue expressed the E-cadherin gene and protein. Of the renal cancer tissues analyzed, 67% (23 of 34) lacked E-cadherin expression, with an associated increase in methylation, compared with normal tissue. E-cadherin gene promoter was methylated in all renal cancer cell lines and was accompanied by a loss of E-cadherin gene and protein expression. The treatment of renal cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine restored E-cadherin mRNA expression in all renal cancer cell lines. This is the first report that shows inactivation of the E-cadherin gene and protein in renal cell carcinoma through CpG hypermethylation in the promoter region of this gene. The results of these experiments may contribute to an understanding of the role of E-cadherin inactivation in renal cell carcinoma.

Frequently deleted loci on chromosome 9 may harbor several tumor suppressor genes in human renal cell carcinoma.

PURPOSE: Loss of various loci on chromosome 9 has been reported in various cancers. To determine the frequency of deletions at different loci of chromosome 9 in renal cell carcinoma microdissected samples of normal renal epithelium and carcinoma from the same patients were analyzed. MATERIALS AND METHODS: DNA was isolated from microdissected sections of normal and tumor cells of 60 renal specimens, amplified by polymerase chain reaction and analyzed for loss of heterozygosity on chromosome 9 using the 16 microsatellite markers D9S178, D9S157, D9S274, D9S168, D9S285, D9S156, D9S1839, D9S162, IFNA, D9S736, D9S171, D9S1749, D9S273D9S270, D9S153 and D9S170. Loss of heterozygosity was analyzed by a polymerase chain reaction based technique developed at our laboratory. RESULTS: This study showed a high incidence of loss of heterozygosity on chromosome 9 in renal cell carcinoma. Of 60 cases 44 (73%), 24 (40%) and 14 (23%) showed loss of heterozygosity at a minimum of 1, at a minimum of 3 and at 4 or more loci, respectively. The main deletion was found on the 9p21 region at loci DS171 in 38% of cases, D9S1749 in 42% and DS270 in 14%. Overall deletion on chromosome 9p21 was noted in 57% of renal cancer cases. Other deleted regions were on chromosome 9p'0022 to 23 at loci D9S157 in 37% of cases, D9S274 in 20%, D9S168 in 27%, D9S285 in 20%, D9S156 in 12%, D9S1839 in 17% and D9S162 in 24%. Overall deletion at chromosome 9q32 to 33 was noted in 46% of renal cell carcinoma cases. Chromosome 9q32 to 33 also showed deletion at locus D9S170 in 22% of renal cell carcinoma cases. When we compared the incidence of deletion at various loci on chromosome 9 according to renal cell carcinoma grade, we found a higher rate of deletion in advanced grades of renal cell carcinoma. A candidate target tumor suppressor gene, p16 (MTS-1/CDKN2), has been identified within the 9p21 deleted region in various cancers. In our study the expression of p16 protein was absent or low in renal cell cancer samples, suggesting that loss of the p16 gene may be involved in renal cell carcinogenesis. CONCLUSIONS: Our study demonstrates a high incidence of loss of heterozygosity on chromosome 9, mainly 9p21 and 9p22 to 23, in renal cell carcinoma, suggesting several putative tumor suppressor genes on these regions. The identification of other tumor suppressor genes on the 9p21 and 9p22 to 23 regions warrants further studies.

Methylation of the E-cadherin gene promoter correlates with progression of prostate cancer.

PURPOSE: We studied the methylation status of E-cadherin gene promoter in prostate cancer and its relationship with E-cadherin inactivation in prostate cancer. MATERIALS AND METHODS: Seven human prostate cell lines and 35 microdissected prostate cancer specimens were analyzed for E-cadherin promoter methylation using the bisulfite genome sequencing technique. E-cadherin messenger (m)RNA expression and protein expression were also studied in prostate cell lines by reverse transcriptase-polymerase chain reaction and in prostate cancer specimens by immunostaining, respectively. RESULTS: The overall methylation of E-cadherin promoter was evident in 14 of 20 grades III to V (70%) and in 5 of 15 grades I to II (33%) prostate cancer samples. It correlated with absent or reduced E-cadherin immunostaining. Methylation in low grade tumors was present mainly in the exon region, whereas in high grade tumors methylation was also present in the promoter region. Methylation was noted in 2 of 6 prostate cancer cell lines (33%) and correlated well with decreased E-cadherin mRNA in these cell lines. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored E-cadherin mRNA levels in the E-cadherin negative prostate cancer cell lines TSUPr1 and DuPro. CONCLUSIONS: Methylation of the E-cadherin gene is common in prostate cancer and the severity of E-cadherin methylation correlates with tumor progression. This study implies that the invasion and metastasis suppressor function of E-cadherin may often be compromised in human prostate cancer by epigenetic rather than by mutational events.

Genital trauma due to animal bites.

PURPOSE: Animal bites to the external genitalia are rare. We retrospectively evaluated our experience with treating genital trauma caused by animal attacks. MATERIALS AND METHODS: We studied the medical records of 10 patients treated in the surgical emergency department at our hospital who presented with genital injury caused by an animal bite from 1983 to 1999. Special attention was given to the severity of injury, surgical treatment, antibiotic prophylaxis and outcome. RESULTS: Of the 2 men and 8 boys 8 were attacked by dogs, 1 by a horse and 1 by a donkey, respectively. In all cases initial local treatment involved débridement and copious wound irrigation with saline and povidone-iodine solution. Five patients who presented with minimal or no skin loss underwent primary skin closure, including 2 in whom urethral lacerations were surgically repaired. There was moderate to extensive tissue loss in 5 patients, including degloving penile injury in 2, traumatic spermatic cord amputation in 1, complete penile and scrotal avulsion in a 5-month-old infant, and partial penectomy in 1. Reconstructive procedures provided satisfactory cosmetic and functional results in 8 cases. Antibiotic prophylaxis was administered in all patients and no infectious complications developed. CONCLUSIONS: Animal bite is a rare but potentially severe cause of genital trauma and children are the most common victims. Morbidity is directly associated with the severity of the initial wound. Because patients tend to seek medical care promptly, infectious complications are unusual. Management involves irrigation, débridement, antibiotic prophylaxis, and tetanus and rabies immunization as appropriate as well as primary wound closure or surgical reconstruction. Good functional and cosmetic results are possible in the majority of cases.

Induction of acute and chronic pancreatitis with the use of the toxin of the scorpion Tityus serrulatus: experimental model in rats.

We observed that the purified venom of the Tityus serrulatus scorpion (T1 fraction), injected i.v. in rats, in a single dose of 0.5 mg/kg, produces: acute pancreatitis, characterized by degranulation and acinar cell vacuolization, necrosis and an inflammatory reaction, 24, 48 and 96 hours after the injection; chronic pancreatitis, characterized by interstitial fibrosis, lymphocyte infiltration, ductal and ductular dilation, acinar cell atrophy, periductal ductular hyperplasia, 20 days after injection: hyperplasia of Langerhans' islets and nesidioblastosis, associated to chronic pancreatitis. The absence of deaths in the experimental group is an interesting finding: the dose used preserved the animals from death and allowed the safe follow-up of the progression of the provoked pancreatitis. The results led us to conclude that the toxin of Tityus serrulatus scorpion is an agent of considerable efficacy in the induction of pancreatitis in rats providing an experimental model of acute and chronic form of this disease.

Nesidioblastosis associated with chronic experimental pancreatitis produced by a scorpion toxin, tityustoxin, in rats.

Tityustoxin (TsTx), the purified venom of the Brazilian scorpion Tityus serrulatus, was injected intravenously (50 micrograms/kg) into rats, producing a typical picture of chronic pancreatitis after 20 days. Nesidioblastosis, a lesion characterized by hyperplasia of the islets of Langerhans, was also detected in a high percentage (40%) of animals. TsTx-induced pancreatitis may be a useful model for the study of nesidioblastosis in laboratory animals.

Nesidioblastosis associated with chronic experimental pancreatitis produced by a scorpion toxin, tityustoxin, in rats

Tityustoxin (TsTx), the purified venom of the Brazilian scorpion Tityus serrulatus, was inected intravenously (50 microng/Kg) into rats, producing a typical picture of chronic pancreatitis after 20 days. Nesidioblastosis, a lesions characterized by hyperplasia of the islets of Langerhans, was also detected in a high percentage (40%) of animals. TsTx-induced pancreatitis may be a useful model for the study of nesidioblastosis in laboratory animals