p16 is a major inactivation target in hepatocellular carcinoma

BACKGROUND. The p16(INK4A) gene encodes 2 cell, cycle regulator proteins, p 16 and p14(ARF) by alternative splicing. This genetic locus also contains a nother cell cycle regulator gene, p15(INK4B), which encodes p15. The inacti vation of the p16 protein has been demonstrated in some hepatocellular carc inomas (HCCs); however, the inactivation of the other 2 cell regulator prot eins and their inactivation patterns are not well characterized. METHODS. To characterize the role of the above 3 cell cycle regulator prote ins in HCCs, the authors examined the genomic status of the p16(INVK4A) and p15(INK4B) genes and their RNA products in 20 HCC tissues and 7 human HCC cell lines. Homozygous deletions in each exon of p16(INK4A) and p15(INK4B) were evaluated by comparative multiplex polymerase chain reaction (PCR), an d the methylation status of the p16(INK4A) and p15(INK4B) promoter region w as analyzed by methylation specific PCR. RESULTS. Homozygous deletions were found in 6 of 20 HCCs (30%) and 2 of 7 H CC cell lines (29%). In 20 HCCs, the frequency of homozygous deletions was 20% in exon 1 of p15(INK4B), 20% in exon 2 of p15(INK4B), 10% in exon Ip of p16(INK4A) 25% in exon 1 alpha of p16(INK4A), 15% in exon 2 of p16(INK4A), and 15% in exon 3 of p16(INK4A). The authors found hypermethylation of the p16(INK4A) promoter region in 7 HCCs (35%) and 3 HCC cell lines (43%). The overall frequency of p16 alterations in HCCs, including hypermethylation a nd homozygous deletions, was 60% (12 of 20 cases). According to reverse tra nscriptase-PCR analysis, the absence of RNA expression was most frequent in p16 (11 of 20 cases, 55%) and less frequent in p15 (7 of 20 cases, 35%) an d p14(ARF) (5 Of 20 cases, 25%). CONCLUSIONS. Among the 3 cell cycle regulator proteins encoded at the 9p21 genetic locus, inactivation of p16 is the most frequent event in HCCs in wh ich promoter hypermethylation and homozygous deletions are the common mecha nisms. (C) 2000 American Cancer Society.