| Research Abstract |
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.
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