RB PROTEIN ALTERATION IN HEPATOCELLULAR CARCINOMA

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Preliminary studies have shown that mutations of the RB tumor suppressor gene can be found in some HBV related human hepatocellular carcinomas (HCC); however, the locations of these mutations within the RB genome have not been determined. In the present study, abnormalities of the RB gene were analyzed within exons 12-23 by single-strand conformation polymorphism (SSCP) using five overlapping segments of 129 to 337 nucleotides each. Five cell lines derived from human HCCs were studied. (All mutations or deletions of the RB gene reported in other human cancers have encompassed portions of exons 12-23.). Total cellular RNA was used to synthesize cDNA; cDNA fragments were then amplified by polymerase chain reaction (PCR) using p-labeled primers and analyzed by SSCP. Deletion in the RB cDNA were found in exons 17-21 in cell lines HLE and HLF, and in exons 20-21 in cell line Hep3B. The exons examined were normal in cell lines PLC/PRF/5 and HUH-7. These deletions (or splice-site mutations with exon-skipping) in the RB gene appear to play a role in hepatocarcinogenesis in some cases, as observed in three of these cell lines. Two of the cell lines appeared to have normal exons 12- 23, although a heterozygous deletion or exon-skipping has not yet been ruled out. The apparent lack of alterations of RB in these two cell lines may reflect the heterogeneity of tumor development. The abnormalities of RB gene does not appear to be correlated with the presence or the absence of integrated HBV-DNA in these cell lines.