p16 is a major inactivation target in hepatocellular carcinoma.
BACKGROUND
The p16(INK4A) gene encodes 2 cell cycle regulator proteins, p16 and p14(ARF), by alternative splicing. This genetic locus also contains another cell cycle regulator gene, p15(INK4B), which encodes p15. The inactivation of the p16 protein has been demonstrated in some hepatocellular carcinomas (HCCs); however, the inactivation of the other 2 cell regulator proteins and their inactivation patterns are not well characterized.


METHODS
To characterize the role of the above 3 cell cycle regulator proteins in HCCs, the authors examined the genomic status of the p16(INK4A) 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), and the methylation status of the p16(INK4A) and p15(INK4B) promoter region was analyzed by methylation specific PCR.


RESULTS
Homozygous deletions were found in 6 of 20 HCCs (30%) and 2 of 7 HCC 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 1beta of p16(INK4A), 25% in exon 1alpha 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 and homozygous deletions, was 60% (12 of 20 cases). According to reverse transcriptase-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%) and 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 which promoter hypermethylation and homozygous deletions are the common mechanisms.
Jin
M
M
Department of Pathology, Brain Korea 21 Projects for Medical Sciences, Yonsei University, College of Medicine, Seoul, Korea.
Piao
Z
Z
Kim
N G
NG
Park
C
C
Shin
E C
EC
Park
J H
JH
Jung
H J
HJ
Kim
C G
CG
Kim
H
H
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D018797:Cell Cycle Proteins; D002471:Cell Transformation, Neoplastic; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D017353:Gene Deletion; D019942:Genes, p16 / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D008745:Methylation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D025803:Tumor Suppressor Protein p14ARF; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D011506:Proteins; D014157:Transcription Factors; D025803:Tumor Suppressor Protein p14ARF; D025521:Tumor Suppressor Proteins; D012313:RNA