Analyses of RB pathway molecules in human hepatocellular carcinoma.s.

人肝细胞癌中RB通路分子的分析。

基本信息

批准号：
11670497
负责人：
NISHIDA Naoshi
金额：
$ 2.24万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670497/
关键词：
RB gene p16 gene cyclin D1 gene p53 gene ARF gene hepatocellular carcinoma metastatic recurrence cell cycle 肝癌 RB経路 p53経路 p16^<INK4A>遺伝子転移再発分化度 PB遺伝子プロモーターメチル化

项目摘要

RB pathway is known to play an important role in regulating cell cycle control. In the present study, we analyzed the aberration of RB pathway molecules comprehensively by using human hepatocellular carcinoma (HCC) tissues to know the role of disruption of RB pathway in hepatocarcinogenesis. In addition, we also study the alteration of p53 and p14ARF and examine the relationship between disruption RB and p53 pathway in HCC.We analyzed the expression of RB with immunohistochemistry (HIS), amplification and overexpression of the cyclin D1 gene with Southern blotting and HIS, alteration and expression of the p16 gene with methylation specific-PCR (MS-PCR), PCR-SSCP, multiplex PCR, and Western blotting. Alteration and expression of p53 was also examined with PCR-SSCP and HIS, and alteration of the p14ARF gene with MS-PCR, PCR-SSCP, multiplex PCR and Taq-Man PCR in the same serious of HCCs.Overexpression of the cyclin D1 was associated with differentiation of HCC, however we could not find any association between alterations of RB/p16 and differentiation of tumor. Alteration of RB/p16 was frequently detected even in well-differentiated HCCs. On the other hand, alteration of p53 was more frequently detected in moderately/poorly-differentiated HCCs than well-differentiated. Mutation of p14ARF, which was accompanied by loss of expression, was found in 7 % of cases. Abnormal methylation in promoter of the p14ARF was not detected in any cases. There was no association between RB and p53 pathway alterations in HCC. To examine the risk of the disruption of RB pathway in metastatic recurrence of HCC, we performed multivariate analysis by using various factors such as size, number, differentiation and stage of tumor with curatively operated HCC cases. However, no association was observed between presence of RB pathway disruption and emergence of metastatic recurrence. These results suggest that abrogation of RB pathway take place in early stage in HCC formation.

已知RB途径在调节细胞周期控制中发挥重要作用。本研究利用人肝细胞癌（HCC）组织对RB通路分子的畸变进行全面分析，以了解RB通路破坏在肝癌发生中的作用。此外，我们还研究了p53和p14ARF的改变，并检查了HCC中RB和p53通路中断之间的关系。我们用免疫组织化学（HIS）分析了RB的表达，用Southern blotting和HIS分析了cyclin D1基因的扩增和过表达。通过甲基化特异性 PCR (MS-PCR)、PCR-SSCP、多重 PCR 和蛋白质印迹分析 p16 基因的改变和表达。还用 PCR-SSCP 和 HIS 检测了 p53 的改变和表达，并用 MS-PCR、PCR-SSCP、多重 PCR 和 Taq-Man PCR 检测了同一严重 HCC 中 p14ARF 基因的改变。 RB/p16 的改变与 HCC 的分化有关，但我们没有发现 RB/p16 的改变与肿瘤的分化之间存在任何关联。即使在分化良好的 HCC 中也经常检测到 RB/p16 的改变。另一方面，p53 的改变在中/低分化 HCC 中比在高分化 HCC 中更常见。 7% 的病例发现 p14ARF 突变并伴有表达缺失。在任何情况下均未检测到 p14ARF 启动子中的异常甲基化。 HCC 中 RB 和 p53 通路改变之间没有关联。为了检查 HCC 转移性复发中 RB 通路中断的风险，我们对经过根治性手术的 HCC 病例使用肿瘤大小、数量、分化和分期等各种因素进行多变量分析。然而，没有观察到 RB 通路破坏的存在与转移复发的出现之间存在关联。这些结果表明，RB 途径的废除发生在 HCC 形成的早期阶段。