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p16 is a major inactivation target in hepatocellular carcinoma
1Department of Pathology, Brain Korea 21 Projects for Medical Sciences, Yonsei University, College of Medicine, Seoul, Korea.
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.
Insights
In hepatocellular carcinoma (HCC), p16 inactivation is frequent due to promoter hypermethylation and homozygous deletions. This study characterizes alterations in p16, p15, and p14(ARF) cell cycle regulators in HCC.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p16(INK4A) gene produces p16 and p14(ARF) proteins; the p15(INK4B) gene produces p15. These are cell cycle regulators.
- While p16 protein inactivation is known in some hepatocellular carcinomas (HCCs), the roles and inactivation patterns of p15 and p14(ARF) are less understood.
Purpose of the Study:
- To investigate the genomic status and RNA expression of p16(INK4A), p15(INK4B), and p14(ARF) in HCC.
- To determine the frequency and mechanisms of inactivation for these cell cycle regulators in HCC.
Main Methods:
- Examined genomic DNA and RNA from 20 HCC tissues and 7 HCC cell lines.
- Utilized comparative multiplex polymerase chain reaction (PCR) to assess homozygous deletions in p16(INK4A) and p15(INK4B) exons.
- Analyzed promoter methylation status using methylation-specific PCR and RNA expression via reverse transcriptase-PCR.
Main Results:
- Homozygous deletions were observed in 30% of HCC tissues and 29% of cell lines.
- Hypermethylation of the p16(INK4A) promoter occurred in 35% of HCCs and 43% of cell lines.
- Overall p16 alterations (deletions, hypermethylation) reached 60%; absence of RNA expression was most common for p16 (55%), followed by p15 (35%) and p14(ARF) (25%).
Conclusions:
- Inactivation of p16 is the most frequent event among the three cell cycle regulators at the 9p21 locus in HCC.
- Promoter hypermethylation and homozygous deletions are the primary mechanisms driving p16 inactivation in HCC.