Published papers

Purpose: DNA methylation is a key regulator of gene transcription and genomic stability, and alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. We elucidated the significance of aberrant protein expression of DNA methyltransferase (DNMT) 1, a major enzyme involved in the determination of genomic methylation patterns, during human urothelial carcinogenesis. Materials and Methods: A total of 61 samples of normal urothelium, 89 noncancerous urothelium. samples showing no remarkable histological changes from patients with bladder cancer (NBCs), 78 dysplastic urothelium samples and 174 transitional cell carcinoma samples (TCCs) were subjected to immunohistochemical analysis for DNMT1. Results: The incidence of nuclear DNMT1 immunoreactivity in NBCs (65%) was significantly higher than in normal urothelium (20%, p <0.0001) and the incidence was even higher in dysplastic urothelium samples (84%, p = 0.0017). The incidence of nuclear DNMT1 immunoreactivity was 87% in TCCs and the intensity of nuclear immunoreactivity was markedly increased in TCCs compared with that in dysplastic urothelium samples. DNMT1 expression levels had already increased in NBCs in which the proliferating cell nuclear antigen labeling index had not yet increased. Increased DNMT1 protein expression correlated significantly with histological grade (p <0.0001). DNMT1 protein expression was higher in nonpapillary tumors (p = 0.0001), especially flat carcinoma in situ, than in papillary tumors. Conclusions: Progressively increasing expression of DNMT1 protein is not entirely a secondary result of increased cell proliferative activity, but rather it is associated with urothelial carcinogenesis even during the precancerous stages. In particular, it is associated with the development of flat carcinoma in situ, which is considered to be a precursor of nodular invasive carcinoma of the bladder.

Purpose: We elucidated the role of methylation in the promoter region of the CDH1 gene in bladder carcinogenesis, particularly in those comprising carcinoma in situ. Materials and Methods: A total of 49 cases of transitional cell carcinoma of the bladder obtained from transurethral resection were examined. Methylation status of the CDH1 promoter region was analyzed by methylation specific polymerase chain reaction from chemically modified DNA after Na-bisulfite treatment. Loss of heterozygosity on 16q was examined by blunt end single strand DNA conformation polymorphism using 4 tetranucleotide repeat microsatellite markers assigned on 16q13 to 22.1. E-cadherin expression was evaluated by immunostaining on formalin fixed, paraffin embedded tissue sections using anti E-cadherin murine monoclonal antibody, HECD1 and standard avidin-biotin immunoperoxidase complex technique. Results: Analysis of the 49 bladder transitional cell carcinoma samples showed CDH1 promoter methylation in 23 (47%). Methylation of the CDH1 gene did not correlate with tumor stage (p = 0.2097) but with high grade transitional cell carcinoma (p = 0.0416). CDH1 promoter methylation was observed at a significantly higher frequency in the carcinoma in situ positive group than in the carcinoma in situ negative group (16 of 18 cases or 89% versus 7 of 31 or 23%, p <0.0001) and it strongly correlated with abnormal E-cadherin expression (p <0.0001). We found 16q loss of heterozygosity in 16 of 47 cases (34%), which correlated with higher histological grade (p = 0.0069) but not with the presence of the carcinoma in situ component (p = 0.1235). Conclusions: This study showed that CDH1 gene promoter methylation is strongly associated with bladder transitional cell carcinoma comprising carcinoma in situ.

Most patients with pancreatic ductal carcinoma have a poor prognosis. However, in certain cases, 5-year survival can be achieved after surgical resection. Analysis of the pathologic findings associated with good survival rates will assist in identifying the optimum treatment. The clinicopathologic features of 67 patients who underwent surgical resection of ductal adenocarcinoma of the pancreas between 1990 and 1996 were reviewed and correlated with survival rates. There were 42 men and 25 women, with a mean age of 62.1 years (range, 44 to 82 years). The mean greatest diameter of the tumor was 4.3 cm (range, 1.5 to 11 cm). Nineteen patients (29.4%) survived more than 3 years, and 9 (13.2%) survived more than 5 years after surgical resection. The intraductal papillary component (IDPC) of the carcinoma was the main focus of the pathologic observations. IDPC was defined as intraductal papillary proliferative lesions seen in the tumor nodule with proliferative cells consistent with carcinomatous cellular atypia. IDPC was clearly present (++) in 24 patients and vaguely present (+) in 9 patients. Using the Mantel-Cox test, a statistically significant correlation was found between the presence of IDPC (either + or ++) and postoperative patient survival (P = .002). IDPC is a morphologic feature associated with longer patient survival and should be taken into consideration in assessing the pathway of tumor progression. HUM PATHOL 32;834-841. (C) 2001 by W.B. Saunders Company.

Aims: Adamantinomatous craniopharyngioma (ACP) resembles histologically some odontogenic tumours, such as ameloblastoma and calcifying odontogenic cyst. However, there has been no evidence that ACP differentiates also functionally as odontogenic epithelium. The aim of this study was to gain evidence of odontogenic epithelial differentiation in ACP by means of immunohistochemistry. Among normal human tissues, enamel proteins are expressed exclusively in teeth, and lymphoid enhancer factor 1 (LEF1), in co-operation with beta-catenin, play an important role in tooth development. The expression of these proteins is therefore indicative of odontogenic epithelial differentiation. Methods and results: The expression of enamel proteins and LEF1 was examined in 10 adamantinomatous and six papillary craniopharyngiomas. All the ACPs showed a variable degree of enamel protein expression, including amelogenin, enamelin and enamelysin, mainly in ghost cells. LEF1 was also heterogeneously expressed in ACPs; remarkably, its expression pattern was identical to that of nuclear beta-catenin accumulation. In contrast, none of the papillary craniopharyngiomas expressed enamel proteins or LEF1. Conclusions: These results suggest that ACP consistently shows odontogenic epithelial differentiation. Since ACPs harbour beta-catenin mutation, the inappropriate activation of beta-catenin/LEF1 complex-dependent transcription may play a critical role in ACP tumorigenesis.

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs), The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarcinogenesis, and that reduced expression of MBD4 may play a role in malignant progression of HCC.

Dysadherin is a membrane glycoprotein expressed strongly in several human cancers; Overexpression of dysadherin in tumor cells is closely associated with malignant phenotype (e.g., metastasis) and poor prognosis. In our analysis,,six pancreatic cancer cell lines showed a positive correlation between dysadherin expression and cell motility. Introduction of small interfering RNA (siRNA) against dysadherin into the Panc-1 cell line caused reduction of dysadherin expression and suppression of cell motility. In contrast, stable transfection of a dysadherin expression vector into the Capan-1 cell line increased cell motility. In vivo, the metastatic potential of orthotopically transplanted Capan-1 tumor cells in severe combined immunodeficient mice was increased by dysadherin overexpression. Cell morphology and actin organization were also influenced by modulation of dysadherin expression. Cells transfected with dysadherin siRNA tended to have a relatively larger, more spread shape and increased transverse actin stress fibers compared with parent cells and cells transfected with control siRNA. Our study suggests that dysadherin is able to modulate actin structures, stimulate cell motility, and contribute directly to the metastatic potential of human pancreatic cancer cells.

Purpose: DNA methylation has important roles in genomic stability. Accordingly DNA hypomethylation on pericentromeric satellite regions may induce chromosomal instability through heterochromatin decondensation and chromosomal recombination enhancement. We elucidated the significance of aberrant DNA methylation on pericentromeric satellite regions during urothelial carcinogenesis. Materials and Methods: We examined DNA methylation status on satellites 2 and 3 by Southern blotting and determined the allelic status of chromosome 9 using 6 microsatellite markers (D9S775, D9S925, D9S304, D9S303, D9S283 and D9S747) in 27 transitional cell carcinomas of the bladder, ureter or renal pelvis and corresponding noncancerous tissues. Results: DNA hypomethylation on satellites 2 and 3 was detected in 2 (7%) and no (0%) noncancerous tissues, and in 11 (41%) and 12 (44%) urothelial carcinomas, respectively. DNA hypomethylation in urothelial carcinomas significantly correlated with histological grade (p = 0.0012 and 0.0043), invasion depth (p = 0.0055 and 0.0228) and morphological structure (papillary vs nodular, p = 0.0161 and 0.0297) for satellites 2 and 3, respectively. Loss of heterozygosity on at least 1 locus of chromosome 9 was detected in 14 urothelial carcinomas (52%). DNA hypomethylation on satellites 2 (p = 0.0098) and 3 (p = 0.0034) significantly correlated with loss of heterozygosity on chromosome 9. Conclusions: DNA hypomethylation on pericentromeric satellite regions may participate in the development and progression of urothelial carcinomas by inducing loss of heterozygosity on chromosome 9.

Purpose: We elucidated the significance of aberrant DNA methylation on multiple CpG islands and its correlation with DNA methyltransferase DNMT1 protein expression during urothelial carcinogenesis. Materials and Methods: We examined the DNA methylation status on multiple CpG islands by methylation specific polymerase chain reaction and combined bisulfite restriction enzyme analysis in 12 specimens of normal urothelium, 23 of noncancerous urothelium showing no remarkable histological changes obtained from patients with bladder cancer (NBC) and 70 of transitional cell carcinoma (TCC). Results: DNA methylation on CpG islands of the p16 (0%, 17% and 21%) and death-associated protein kinase (13%, 33% and 29%) genes, and methylated in tumor-2 (56%, 60% and 76%), 12 (0%, 6% and 30%), 25 (25%, 27% and 35%) and 31 (45%, 56% and 79%) clones was detected in normal urothelium, NBCs and TCCs, respectively. The incidence of concurrent DNA hypermethylation on 3 or more CpG islands in NBCs (38%) was significantly higher than that in normal urothelium (0%, p = 0.0455) and even higher in TCCs (59%, p = 0.0043). The incidence of the CpG island methylator phenotype in nonpapillary carcinomas (nodular invasive carcinomas and their precursors, ie flat carcinoma in situ, 71%) was significantly higher than in papillary carcinomas (40%, p = 0.0143). In all specimens examined concurrent DNA hypermethylation on 3 or more CpG islands significantly correlated with immunohistochemically evaluated DNMT1 protein over expression (p = 0.0167). Conclusions: DNA hypermethylation on multiple CpG islands in association with DNMT1 protein over expression may participate in multistage urothelial carcinogenesis even at the precancerous stage and particularly in the development of nodular invasive carcinomas of the bladder.

BACKGROUND. The E-cadherin-mediated cell adhesion system is frequeutly inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently, the authors reported a novel cell membrane glycoprotein, dysadherin, which has an anti-cell-cell adhesion function and down-regulates E-cadherin. METHODS. Expression of both dysadherin and E-cadherin was investigated immunohistochemically in 115 patients with cutaneous malignant melanoma to determine the correlation between the 2 molecules and their associations with both patient survival and the clinicopathologic features of the tumors. RESULTS. Dysadherin and E-cadherin were expressed at the cell membranes of melanoma cells. Fifty-two percent of the tumors showed dysadherin immunopositivity, and 91% of the tumors showed reduced E-cadherin immunopositivity. There was no significant inverse correlation between dysadherin expression and E-cadherin expression. Increased dysadherin expression was significantly correlated with nodular subtype (P = 0.042), Clark level (P < 0.001), tumor thickness (P < 0.001), ulceration (P = 0.008), lymph node metastasis (P < 0.001), high TNM classification (P < 0.001), and poor patient survival (P < 0.001). Multivariate analysis of patient survival revealed that increased dysadherin expression was a significant predictor of poor Survival (P < 0.001). CONCLUSIONS. Thus, increased expression of dysadherin was a significant indicator of poor prognosis in patients with cutaneous malignant melanoma. (c) 2005 American Cancer Society.

In a search for signalling molecules that act downstream of E-cadherin inactivation in cancer, we examined the expression and localization of E-cadherin-associated proteins in lobular carcinoma, in which the E-cadherin gene is frequently inactivated, and found that E-cadherin down-regulation correlated with the cytoplasmic localization of p120ctn. Similar cytoplasmic localization of p120ctn and growth factor-induced accumulation of tyrosine-phosphorylated p120ctn in the protrusive domain were observed in E-cadherin-deficient breast cancer cells. Down-regulation of endogenous p120ctn by RNA interference promoted stress fiber formation and induced a flattened morphology with an increase of Rho-GTPase activity; it also reduced the development of membranous protrusions and migratory activity in E-cadherin-deficient breast cancer cells. Inactivation of E-cadherin in cancer cells is associated with the conversion from epithelial to mesenchymal phenotype, which also occurs in physiological conditions such as developmental processes. Cytoplasmic localization of p120ctn accompanied by E-cadherin down-regulation was observed in mesoderm cells that had undergone epithelial-mesenchymal transition during early mouse embryogenesis. Collectively, our results suggest that cytoplasmic p120ctn may contribute to the invasive phenotype of E-cadherin-deficient breast cancer cells.

We present a case of collecting duct carcinoma (CDC) of the kidney exhibiting uncommon radiologie findings. A 67-year-old man had a history of hepatic resection for hepatocellular carcinomas. Follow-up CT revealed a left renal mass that presented an exophytic appearance with distortion of the renal contour. Since malignancy was suspected, the patient underwent a left radical nephrectomy with partial adrenalectomy and para-aortic lymph nodes dissection. Histologically, the majority of the tumor showed a tubular structure, and the rest was a solid area resembling transitional cell carcinoma (TCC). A diagnosis of CDC was made. In previous reports, common radiologie findings of CDC have been a medullary location and infiltrative appearance. Therefore, the outer contour of the affected kidney is usually maintained when the mass is relatively small. However, the present case did not show these common findings. In conclusion, the radiologic findings of CDC may be various and we should consider this tumor in the differential diagnosis of renal tumors. © 2003 Elsevier Ireland Ltd. All rights reserved.

Changes in DNA methylation status are not only important for regulating gene expression but are also suggested to induce chromosome instability. To reveal the correlation of DNA methylation status in heterochromatin regions with tumor histology and with chromosome alterations, DNA methylation status was examined by Southern blot analysis, and numerical and structural chromosome alterations, including the formation of der(16)t(1;16)/der(1;16), were examined by fluorescence in situ hybridization at the two loci in the pericentromeric satellite 2 regions of chromosomes 16 and 1 in 39 human breast carcinomas. DNA hypomethylation at the D16Z3 and the D1Z1 loci was detected in 31% (12 of 39) and 36% (12 of 33) of carcinomas, respectively, and mostly concurred. DNA hypomethylation was more frequent in the carcinoma group of more aggressive histological types or grade 3 than in the carcinoma of less aggressive histological types or grades 1 and 2, and tended to be more frequent in carcinomas with greater than or equal to4 copies of chromosomes 16 and/or 1 than in carcinomas with less than or equal to3 copies of any of these chromosomes. The frequency of DNA hypomethylation at the D16Z3 and the D1Z1 loci was 45% (10 of 22) and 53% (9 of 17) in carcinomas without der(16)t(1;16)/der(1;16), formation, but only 12% (2 of 17) and 19% (3 of 16) in carcinoma with der(16)t(1;16)/der(1;16), respectively (P = 0.036 and 0.070). The 16q breakage was almost equally detected between carcinoma groups with and without the DNA hypomethylation. DNA hypomethylation in the satellite 2 regions was suggested to be associated with the accumulation of a large number of numerical chromosome alterations and involved in the development of breast carcinomas of aggressive histological features. on the contrary, chromosome instability induced by mechanisms other than DNA hypomethylation in heterochromatin regions might cause the formation of der(16)t(1;16)/der(1;16) and less aggressive breast carcinomas.

Purpose: Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, correlates significantly with DNA hypomethylation in pericentromeric satellite regions, which is known to result in centromeric decondensation and enhanced chromosomal recombination in precancerous conditions and hepatocellular carcinomas (HCCs). We aimed to elucidate further the significance of DNMT3b4 during human hepatocarcinogenesis. Methods: DNMT3b4-transfected human epithelial 293 cells were characterized using growth rate measurements, gene expression microarray, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR was also performed on eight normal liver specimens, 45 noncancerous liver specimens showing chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 56 HCCs. Results: The growth rate of the DNMT3b4 transfectants was about double that of mock-transfectants. Induction of signal transducer and activator of transcription 1 (STAT1), an effector of interferon signaling, and of a set of downstream genes implicated in such signaling, was observed in the DNMT3b4 transfectants. There was significant correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs. mRNA expression levels of STAT1 and the three downstream genes examined were all significantly elevated in the chronic hepatitis and cirrhosis specimens compared with the normal liver specimens. Among the HCCs, the mRNA expression levels of STAT1 and the downstream genes were higher in tumors without portal vein involvement than in more malignant HCCs with portal vein involvement. Significant correlations between the mRNA expression levels of STAT1 and each of the downstream genes were observed in the tissue samples. Conclusions: Overexpression of DNMT3b4 is involved in human hepatocarcinogenesis, even at the precancerous stages, not only by inducing chromosomal instability but also by affecting the expression of specific genes.