研究者詳細 - 坂元　亨宇

論文 - 坂元　亨宇

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肝炎と肝癌の分子病理学

坂元亨宇

Molecular Medicine 1139 （ 8 ） 2002年

研究論文（学術雑誌）, 単著

肝発癌の分子機構

金井弥栄，坂元亨宇

Annual Review消化器 2002 113-118 2002年

研究論文（学術雑誌）, 共著

Overexpression of tetraspanin CO-029 in hepatocellular carcinoma

Kanetaka, K., Sakamoto, M., Yamamoto, Y., Yamasaki, S., Lanza, F., Kanematsu, T. and Hirohashi, S.

Journal of Hepatology 35 （ 5 ） 637 - 642 2001年11月

ISSN 0168-8278

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Involvement of c-Src in carcinoma cell motility and metastasis

Sakamoto, M., Takamura, M., Ino, Y., Miura, A., Genda, T. and Hirohashi, S.

Japanese Journal of Cancer Research 92 （ 9 ） 941 - 946 2001年09月

ISSN 0910-5050

Carcinoma cells exhibit dysfunction/dysregulation of cell adhesion systems that correlates with their abilities to migrate, invade, and metastasize. Here we show that the tyrosine kinase c-Src is required for motility and metastasis of two carcinoma cell lines. Adherent KYN-2 cells having a high level of c-Src kinase activity become scattered, extend lamellipodia, and exhibit high motility. Expression of a dominant-negative mutant form of c-Src caused formation of stress fibers and focal adhesions, and markedly reduced motility. HCT15 cells extended lamellipodia and became scattered in response to lysophosphatidic acid stimulation in parallel with transient activation of c-Src, which was inhibited by expression of a dominant-negative mutant form of c-Src or treatment with a specific Src kinase inhibitor. Furthermore, implantation of dominant-negative c-Src transfectants into the peritoneal cavity of SCID mice resulted in reduced peritoneal dissemination compared with control transfectants. These findings indicate that c-Src activation is critically involved in carcinoma cell migration and metastasis.

Inhibition of intrahepatic metastasis of human hepatocellular carcinoma by Rho-associated protein kinase inhibitor Y-27632

Takamura, M., Sakamoto, M., Genda, T., Ichida, T., Asakura, H. and Hirohashi, S.

Hepatology 33 （ 3 ） 577 - 81 2001年03月

ISSN 0270-9139

Intrahepatic metastasis is one of the most important prognostic factors for patients with hepatocellular carcinoma (HCC). Cell motility mediated by Rho- and p160 Rho-associated coiledcoil forming protein kinase (p160ROCK) signaling pathways has recently been shown to play a critical role in intrahepatic metastasis in human HCC. Furthermore, the stable introduction of dominant-negative p160ROCK into Li7 cells resulted in a reduced metastatic rate in mice with severe combined immunodeficiency (SCID). To investigate whether the specific p160ROCK inhibitor, Y-27632, could also inhibit intrahepatic metastasis, the effect of Y-27632 on the cell motility and intrahepatic metastasis of Li7 was investigated. Y-27632 markedly blocked actin reorganization and motility of Li7 cells mediated by lysophosphatidic acid (LPA). Y-27632 was administered continuously into the peritoneal cavity using a micro-osmotic pump, together with orthotopic implantation of Li7 cells into the liver of SCID mice. Phosphate-buffered saline (PBS) alone was administered as the control. The incidence of mice with metastatic nodules decreased in the Y-27632-treated group. The primary tumor volume at the site of injection was smaller in the Y-27632-treated group compared with the control group, but the difference was not statistically significant. Histologically, control tumors showed infiltrative growth into the sinusoidal area at the tumor boundary, whereas Y-27632-treated tumors showed expansive growth and low invasiveness. These findings confirm the importance of the Rho/p160ROCK signaling pathway in intrahepatic metastasis of human HCC, and indicate that Y-27632 may be useful for the prevention of intrahepatic metastasis of human HCC.

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Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis

Saito Yoshimasa, Kanai Yae, Sakamoto Michiie, Saito Hidetsugu, Ishii Hiromasa, Hirohashi Setsuo

Hepatology 33 （ 3 ） 561-568 2001年03月

研究論文（学術雑誌）, 共著

Cloning and characterization of a novel gene, DRH1, down-regulated in advanced human hepatocellular carcinoma

Yamamoto, Y., Sakamoto, M., Fujii, G., Kanetaka, K., Asaka, M. and Hirohashi, S.

Clinical Cancer Research 7 （ 2 ） 297 - 303 2001年02月

ISSN 1078-0432

Few genes related to carcinogenesis and progression of hepatocellular carcinoma (HCC) have been identified to date. In the present study, we report the cloning and characterization of a novel gene, DRH1, which is frequently down-regulated in HCC. The full-length DRH1 clone contains an open reading frame of 1257 nucleotides encoding 419 amino acids. The deduced DRH1 protein shows 41% identity to VDUP1, expression of which is rapidly induced by 1,25-dihgdroxyvitamin D-3. The DRH1 gene was localized to chromosome 15, and DRH1 protein was mainly observed in the cytoplasm of transiently transfected cells. Real-time quantitative reverse transcription-PCR analysis showed that the expression level of DRH1 was reduced in 29 of 35 (83 %) HCCs compared with corresponding noncancerous liver tissue. The average (mean +/- SE) ratio of DRH1 expression level in tumor to corresponding noncancerous tissue was significantly different between well, moderately, and poorly differentiated HCCs (1.15 +/- 0.23, 0.69 +/- 0.10, and 0.19 +/- 0.04, respectively) and between HCCs without and with vascular invasion (0.93 +/- 0.16 and 0.46 +/- 0.07, respectively). These results indicate that the down-regulation of DRH1 occurs not at an early stage but rather at a late stage of HCC progression. Although the function of DRH1 protein is still unknown, our findings suggest that DRH1 is related to the progression of HCC and may provide a new prognostic factor.

特集：知っておくべき術中診断法肝癌の術中診断-病理-

坂元亨宇

外科 64 （ 1 ） 32-36 2001年01月

研究論文（学術雑誌）, 単著

Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial

Takayama, T., Sekine, T., Makuuchi, M., Yamasaki, S., Kosuge, T., Yamamoto, J., Shimada, K., Sakamoto, M., Hirohashi, S., Ohashi, Y. and Kakizoe, T.

Lancet 356 （ 9232 ） 802 - 807 2000年09月

ISSN 0140-6736

Background Postsurgical recurrence of hepatocellular carcinoma (HCC) is frequent and fatal. Adoptive immunotherapy is active against HCC. We assessed whether postoperative immunotherapy could lower the frequency of recurrence.
Methods Between 1992 and 1995, we did a randomised trial in which 150 patients who had undergone curative resection for HCC were assigned adoptive immunotherapy (n=76) or no adjuvant treatment (n=74). Autologous lymphocytes activated vitro with recombinant interleukin-2 and antibody to CD3 were infused five times during the first 6 months. Primary endpoints were time to first recurrence and recurrence-free survival and analyses were by intention to treat.
Findings 76 patients received 370 (97%) of 380 scheduled lymphocyte infusions (mean cell number per patient 7.1x10(10) [SD 2.1]; CD3 and HLA-DR ceils 78% [16]), and none had grade 3 or 4 adverse events. After a median follow-up of 4.4 years (range 0.2-6.7), adoptive immunotherapy decreased the frequency of recurrence by 18% compared with controls (45% [59] vs 57% [77]) and reduced the risk of recurrence by 41% (95% CI 12-60, p=0.01). Time to first recurrence in the immunotherapy group was significantly longer than that in the control group (48% [37-59] vs 33% [22-43] at 3 years, 38% [22-54] vs 22% [11-34] at 5 years; p=0.008). The immunotherapy group had significantly longer recurrence-free survival (p=0.01) and disease-specific survival (p=0.04) than the control group. Overall survival did not differ significantly between groups (p=0.09).
Interpretation Adoptive immunotherapy is a safe, feasible treatment that can lower recurrence and improve recurrence-free outcomes after surgery for HCC.

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Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells

Genda, T., Sakamoto, M., Ichida, T., Asakura, H. and Hirohashi, S.

Lab Invest 80 （ 3 ） 387 - 94 2000年03月

ISSN 0023-6837

The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell-substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC.

Cell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma

Genda, T., Sakamoto, M., Ichida, T., Asakura, H., Kojiro, M., Narumiya, S. and Hirohashi, S.

Hepatology 30 （ 4 ） 1027 - 36 1999年10月

ISSN 0270-9139

Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasizes to other parts of the liver even at a relatively early stage of the disease, with less tumor spread occurring outside the liver. This intrahepatic metastasis is the main cause of liver failure and death in HCC patients. To analyze the mechanisms of intrahepatic metastasis we have constructed metastatic models using orthotopic implantation of human HCC cell lines. Five HCC cell lines formed liver tumors after injection into the livers of SCID mice, and of those 5 cell lines, Li7 and KYN-2 cells also resulted in vascular tumor thrombi and intrahepatic metastasis. These 2 cell lines had markedly higher cell motilities than the other 3 cell lines in vitro. Their motilities appeared to be Rho-mediated; serum and lysophosphatidic acid (LPA) evoked actin reorganization and motility of Li7 cells, and C3 exoenzyme exposure reduced the motility of both serum-stimulated Li7 cells and KYN-2 cells. Dominant negative and active forms of p160 Rho-associated coiled-coil forming protein kinase (p160ROCK), one of the downstream effectors of Rho, were separately and stably introduced into Li7 cells. Dominant active p160ROCK transfectants showed increased motility that was independent of serum and LPA, and dominant negative p160ROCK transfectants showed reduced motility under stimulation. Furthermore, implantation of dominant negative p160ROCK transfectants resulted in a reduced metastatic rate in vivo compared with the parent cells or a control transfectant. These findings indicate that cell motility mediated by the Rho/p160ROCK signaling pathway plays a critical role in intrahepatic metastasis of human HCC.

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Microsatellite instability associated with hepatocarcinogenesis

Kondo, Y., Kanai, Y., Sakamoto, M., Mizokami, M., Ueda, R. and Hirohashi, S.

J Hepatol 31 （ 3 ） 529 - 36 1999年09月

ISSN 0168-8278

BACKGROUND/AIMS: The biological and clinicopathological significance of microsatellite instability in hepatocellular carcinoma still remains to be determined. The aim of this study was to assess the role of microsatellite instability in hepatocarcinogenesis. METHODS: Genomic DNA extracted from 38 fresh samples of hepatocellular carcinoma was amplified by polymerase chain reaction using 29 fluorescence-labeled microsatellite markers and analyzed using a semi-automated laser scanning system. Associations between the incidence of replication error and the clinicopathological features of hepatocellular carcinoma were evaluated. Since reference DNA was extracted from corresponding fresh samples of non-cancerous liver tissue, the incidence of microsatellite instability in non-cancerous liver tissues was not assessed in this study. RESULTS: Four (11%) hepatocellular carcinomas had a replication error in one or two microsatellite markers; they were all poorly differentiated hepatocellular carcinomas. The incidence of replication error correlated significantly with the histological differentiation of the tumor (p<0.05) and with portal vein involvement (p<0.05). All four hepatocellular carcinomas with replication errors showed loss of heterozygosity in one or more of the 29 markers we examined. No replication errors were detected in six markers in the coding regions of the BAX, insulin-like growth factor II receptor, transforming growth factor-beta type II receptor, E2F-4, hMSH3 and hMSH6 genes. CONCLUSIONS: The results of this study indicate that: (1) microsatellite instability is a rare event during hepatocarcinogenesis and may be specifically associated with progression of hepatocellular carcinoma; and (2) frame-shift mutation in the above six genes is not a common mechanism involved in progression of this cancer.

Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis

Osada, T., Sakamoto, M., Nagawa, H., Yamamoto, J., Matsuno, Y., Iwamatsu, A., Muto, T. and Hirohashi, S.

Cancer 85 （ 4 ） 819 - 31 1999年02月

ISSN 0008-543X

BACKGROUND: The authors previously reported increased ubiquitin (Ub) immunoreactivity in hepatocellular carcinomas (HCCs) and suggested a possible correlation between changes in ubiquitinated protein levels and multistep hepatocarcinogenesis. The current study was performed to identify one of these ubiquitinated proteins (42 kDa) and to analyze the clinical significance of its accumulation. METHODS: The protein was purified using two-dimensional gel electrophoresis and identified by amino acid sequence analysis. The authors studied the expression of this protein in 101 HCCs and 23 precancerous lesions by immunohistochemical methods and in 26 HCCs by immunoblot analysis. A survival analysis was performed on patients with advanced HCC using the Kaplan-Meier method with approximate chi-square statistics for the log rank test. RESULTS: The target protein for ubiquitination was identified as glutamine synthetase (GS). Accumulation of GS was found in 19 of 49 advanced HCCs (38.8%) by immunohistochemical methods and in 9 of 16 (56.3%) by immunoblot analysis, whereas the frequency was much lower in early HCCs (12.9% and 33.3%, respectively) and precancerous lesions (4.3% by immunostaining). In the Ub immunoblot analysis of strongly GS positive specimens, an intense 42-kDa ubiquitinated band was observed. Nine of 21 (42.9%) nodule-in-nodule type HCCs showed a GS positive, high-grade component within a GS negative, low-grade component, indicating the acquisition of GS expression during progression. Among 23 patients with a single advanced HCC nodule, the relapse free survival time was significantly shorter in the GS positive group than in the GS negative group. CONCLUSIONS: The results of this study demonstrate the acquisition of GS expression during hepatocarcinogenesis and the possible regulation of GS enzyme activity by a Ub-dependent proteolytic system. Moreover, GS might play a significant role in promoting the metastatic potential of HCC.

Early hepatocellular carcinoma as an entity with a high rate of surgical cure

Takayama, T., Makuuchi, M., Hirohashi, S., Sakamoto, M., Yamamoto, J., Shimada, K., Kosuge, T., Okada, S., Takayasu, K. and Yamasaki, S.

Hepatology 28 （ 5 ） 1241 - 1246 1998年11月

ISSN 0270-9139

Early hepatocellular carcinoma (HCC) has been defined as a well-differentiated cancer containing Glisson's triad, but it remains unknown whether this lesion is curable. We prospectively studied 70 patients (enrolled from 1,172 referrals between 1982 and 1991) who had a diagnosis of a single HCC 2 cm or less in diameter (Stage T1) and who underwent curative hepatectomy and long-term follow-up (range, 0.2 to 14.3 years), Patients were eligible for surgery if they had a tumor that met the diagnostic criteria for HCC and were in Child-Pugh class A (n = 59) or B (n = 11) status. Among the 70 patients, there was 1 operative death. Based on our typing system, the tumors were assigned as early HCC (n = 15), overt HCC (n = 52), and non-HCC tumor (n = 3), The rate of microscopic regional spread was lower in early HCCs than in overt HCCs (7% vs. 42%; P =.01). The early HCC group had a longer time to recurrence than did the overt HCC group (3.9 vs. 1.7 years; P <.001) and had no local recurrence, After a median follow-up of 6.3 years, both overall survival and recurrence-free survival in the early HCC group were significantly better than those in the overt HCC group (P =.01; P =.001). In these two groups, the 5-year rates of overall survival were 93% and 54% (P =.01), and those of recurrence-free survival were 47% and 16% (P =.05), respectively; a significant survival benefit persisted over a decade (57% vs. 21%; P =.05). The early HCC group was at a lower risk of recurrence (relative risk, 0.31; 95% CI, 0.15 to 0.65; P =.002) and death (relative risk, 0.26; 95% CI, 0.09 to 0.73; P =.01) than was the overt HCC group. Early HCC is a distinct clinical entity with a high rate of surgical cure, thereby justifying its definition. It can be a lesion that corresponds to "Stage 0" cancer in other organs.

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Hyperplastic foci reflect the risk of multicentric development of human hepatocellular carcinoma

Sugitani, S., Sakamoto, M., Ichida, T., Genda, T., Asakura, H. and Hirohashi, S.

J Hepatol 28 （ 6 ） 1045 - 53 1998年06月

ISSN 0168-8278

BACKGROUND/AIMS: Identification of the risk factors of multicentric hepatocarcinogenesis is important for the clinical management of hepatocellular carcinoma. We investigated hyperplastic foci in non-cancerous liver parenchyma, and clarified their pathological features and clinical significance. METHODS: Hyperplastic foci were defined as hypercellular areas, which architecturally and cytologically resembled early hepatocellular carcinoma or adenomatous hyperplasia but did not form macroscopically detectable nodules. Surgically resected livers from 155 patients with hepatocellular carcinoma were examined histopathologically and immunohistochemically. RESULTS: Hyperplastic foci were found in 26 of 155 patients (16.8%). All the patients with hyperplastic foci had chronic liver diseases, and the incidence did not differ between those with chronic hepatitis and those with liver cirrhosis. Six of 92 (6.5%) patients with single primary hepatocellular carcinoma nodules, 8 of 42 (19.0%) with two nodules, and 12 of 21 (57.0%) with more than three nodules had hyperplastic foci. The incidence of hyperplastic foci showed a significant positive correlation with the multiplicity of hepatocellular carcinoma nodules. Immunohistochemically, hyperplastic foci were masses of proliferative hepatocytes similar to adenomatous hyperplasia and early hepatocellular carcinoma. CONCLUSIONS: Hyperplastic foci reflect the risk of multicentric hepatocarcinogenesis. Our results suggest strongly that hyperplastic foci are precursors of adenomatous hyperplasia or hepatocellular carcinoma.

Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver

Osada, T., Sakamoto, M., Nishibori, H., Iwaya, K., Matsuno, Y., Muto, T. and Hirohashi, S.

J Hepatol 26 （ 6 ） 1266 - 73 1997年06月

ISSN 0168-8278

BACKGROUND/AIMS: Ubiquitin covalently attaches to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Increased ubiquitin immunoreactivity was recently observed immunohistochemically in human malignant tumors. To clarify the change in protein metabolism during hepatocarcinogenesis, we studied ubiquitin immunoreactivity in hepatocellular carcinomas (HCCs) and precancerous lesions using immunohistochemistry and immunoblot analysis. METHODS: A total of 72 HCCs (37 advanced, 19 early, 16 early-advanced (advanced HCC component in early HCC nodule) type HCCs) and 18 precancerous lesions (8 atypical adenomatous hyperplasias (AAHs), 10 adenomatous hyperplasias (AHs)) were studied immunohistochemically. Immunoblot analysis was also performed in advanced HCC and early HCC cases. RESULTS: Non-tumorous hepatocytes were either immunonegative or weakly stained in their nuclei. Advanced HCCs showed strong immunoreactivity in most cases, while early HCCs showed relatively weaker immunoreactivity. In 14 of 16 early-advanced type tumors, the inner portion of the nodules, which corresponds to advanced HCC, showed stronger immunoreactivity than the outer low-grade portion. In 8 of 8 AAHs and 7 of 10 AHs, positive but weak staining was found. Immunoblot analysis showed an increase in 42 kDa ubiquitinated protein(s) in 8 of 16 advanced HCC cases (50%) and in 1 of 6 early HCC cases (16.7%), as well as an increase in several other bands in tumor tissues. CONCLUSIONS: The intensity of ubiquitin staining appeared to increase in a stepwise manner from AH to advanced HCC, and the results suggest a possible correlation between changes in the ubiquitinated proteins and multistep hepatocarcinogenesis.

Reduced p21(WAF1/CIP1) expression and p53 mutation in hepatocellular carcinomas

Hui, A. M., Kanai, Y., Sakamoto, M., Tsuda, H. and Hirohashi, S.

Hepatology 25 （ 3 ） 575 - 9 1997年03月

ISSN 0270-9139

p21(WAF1/CIP1) is a universal cyclin-dependent kinase (cdk) inhibitor, the expression of which is regulated by p53-dependent and p53-independent pathways. We examined p21(WAF1/CIP1) expression in and p53 status of 21 primary hepatocellular carcinomas (HCCs) by reverse-transcriptase polymerase chain reaction (RT-PCR) and by PCR single-strand conformation polymorphism (PCR-SSCP) analysis. p21(WAF1/CIP1) messenger RNA expression was reduced markedly in 8 of 21 HCCs (38.1%) and 5 of these 8 HCCs bore p53 mutations. The relative p21(WAF1/CIP1) messenger RNA expression value of HCCs with p53 mutations (.73 +/- .13 U, n = 6) was significantly lower than that of HCCs with wild-type p53 (1.00 +/- 0.21 U, n = 14; P < .01). The p21(WAF1/CIP1) expression levels in cancerous tissues (.73 +/- .13 U) were significantly reduced in comparison with those in noncancerous tissues (.97 +/- .13 U) (P < .01) in the 6 cases with p53 mutations. These data indicate that p21(WAF1/CIP1) expression in HCCs is predominantly regulated by dependence on p53 and that reduced p21(WAF1/CIP1 expression may participate in hepatocarcinogenesis.

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E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma

Osada, T., Sakamoto, M., Ino, Y., Iwamatsu, A., Matsuno, Y., Muto, T. and Hirohashi, S.

Hepatology 24 （ 6 ） 1460 - 7 1996年12月

ISSN 0270-9139

In human hepatocellular carcinoma (HCC), the liver is the major target organ of metastasis, which is known as intrahepatic metastasis. To analyze the mechanism of this metastasis, we established two sublines from the human HCC cell line Li7. Subline Li7HM produced multiple liver metastasis, whereas subline Li7NM never did so after intrasplenic injection into nude mice. Two-dimensional gel electrophoresis and immunoblot analysis showed that only Li7NM expressed vimentin and lacked E-cadherin expression, indicating that this clone had undergone epithelial-mesenchymal transition. We then transfected mouse E-cadherin complementary DNA into Li7NM cells and found that the transfectant cells (EM16.21B.3) formed liver metastasis (8/16 mice) after intrasplenic injection and liver tumors (11/13 mice) after intrahepatic injection, whereas the control cell line formed no tumors. These results suggest that E-cadherin plays an important role in the process of intrahepatic metastasis of HCC.

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Numerical chromosome aberrations in hepatocellular carcinoma detected by fluorescence in situ hybridization

Ohsawa, N., Sakamoto, M., Saito, T., Kobayashi, M. and Hirohashi, S.

J Hepatol 25 （ 5 ） 655 - 62 1996年11月

ISSN 0168-8278

AIMS/METHODS: Numerical aberrations of chromosomes 7,8,9,10,12,17,18, X and Y were examined in 38 hepatocellular carcinoma specimens using centromere-specific DNA probes by the fluorescence in situ hybridization method. RESULTS: Numerical aberrations in more than one of the chromosomes examined were found in 27 of 38 (71%) specimens; 6 of 15 (40%) well-differentiated hepatocellular carcinomas; 15 of 17 (88%) moderately differentiated hepatocellular carcinomas; and all of 6 (100%) poorly differentiated hepatocellular carcinomas. Of 6 early hepatocellular carcinomas, numerical chromosome aberrations were detected in 2. The incidence of numerical chromosome aberrations was 93.8% in patients with portal vein thromboses and/or intrahepatic metastases, 52.4% without portal vein thromboses and/or intrahepatic metastases (p < 0.05), while 89.5% of patients with a tumor more than 3 cm in diameter and 50.0% with a tumor less than 3 cm in diameter had chromosome aberrations (p < 0.05). CONCLUSIONS: These results suggest that numerical chromosome aberrations start to occur in the early stage of hepatocellular carcinoma and to accumulate during tumor progression.

Inactivation of p16INK4 in hepatocellular carcinoma

Hui, A. M., Sakamoto, M., Kanai, Y., Ino, Y., Gotoh, M., Yokota, J. and Hirohashi, S.

Hepatology 24 （ 3 ） 575 - 9 1996年09月

ISSN 0270-9139

We analyzed the p16INK4 status of 6 hepatocellular carcinoma (HCC) cell lines and 32 primary HCC tumors, including 9 early-stage tumors, to determine whether p16INK4 tumor-suppressor gene inactivation participates in hepatocarcinogenesis. p16INK4 was studied at its protein level through Western blotting, at its messenger RNA (mRNA) level through reverse-transcriptase polymerase chain reaction analysis (RT-PCR) and Northern blotting, and at its genomic level through Southern blotting and PCR-single-strand conformation polymorphism analysis. The p16 protein was absent from 3 of 6 cell lines (50%) and 11 of 32 primary tumors (34%), but present in noncancerous tissues, indicating that p16INK4 is involved in hepatocarcinogenesis. Furthermore, we suggest that the p16 protein loss may contribute to the following: (1) early-stage hepatocarcinogenesis, because it was observed in 22% of early stage tumors; and (2) tumor progression, because it occurred approximately twice as often in advanced rather than in early stage tumors (40%). It was striking that neither p16INK4 homozygous deletion and mutation nor loss of p16INK4 mRNA expression were observed in HCC cell lines and primary tumors, including those specimens from which the p16 protein was absent except the Li7HM cell line, in which p16INK4 mRNA was not detected. These results suggest that p16INK4 in HCC is inactivated predominantly by posttranscriptional regulation rather than by genomic aberrations and lack of transcription.

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