Abrogation of the p16-Rb pathway in Korean hepatocellular carcinomas

Abstract

Background/aims: The tumor suppressor gene p16 on human chromosome 9p21 encodes a specific inhibitor of the cyclin D-CDK4 complex which inactivates the Rb protein by hyperphosphorylation. Many reports show that p16 is inactivated in a variety of human cancers. We investigated whether abnormalities involving p16 and Rb are associated with hepatocellular carcinomas in Korea.

Methodology: We performed loss of heterozygosity analysis on 9 primary hepatocellular carcinomas using 7 microsatellite markers spanning human chromosome 9p. Reverse transcriptase-PCR, ribonuclease protection assay, and immunoblotting were used to examine the expression of p16 and Rb in 8 hepatocellular carcinoma cell lines, including 5 from Korean patients. Exons 1 and 2 of the p16 gene were sequenced.

Results: We found a 33% loss of heterozygosity at the D9S171 locus (9p21 region) in the primary hepatocellular carcinomas. The p16 protein was not found in 4 out of 5 (80%) of the Korean cell lines. Among them, 2 cell lines lacked p16 protein and had the following point mutations in p16 exon 2: Asp125 to Asn and Arg58 to Ter. Two of the Korean cell lines and the SK-Hep-1 cells contained deletions in the p16 gene. All cell lines examined, except Hep 3B, expressed Rb protein, which in all cases was dominantly hyperphosphorylated (inactivated).

Conclusions: Our results suggest that p16 and Rb abnormalities are associated with hepatocellular carcinomas in the Korean population.