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Alterations of p16INK4A and p15INK4B genes in gastric carcinomas
1Department of Internal Medicine, Han Yang University School of Medicine, Seoul, Korea.
Background:
It has been suggested that cyclin-dependent kinase inhibitors (CDKIs), including p16 and p15, are tumor suppressor genes. Alterations of CDKIs have been found in most types of cancer. However, little is known about the status of p16 and p15 genes, including methylation of the promoter region, in gastric carcinoma.
Methods:
Thirty-six primary gastric tumors and 9 gastric carcinoma cell lines were examined for alterations of the p16 and p15 genes. Deletion of the p16 and p15 genes was assessed by Southern blot analysis, expression by Northern blot analysis, and mutation by polymerase chain reaction-single strand conformation polymorphism followed by direct sequencing. The methylation status of the 5' CpG island of the p16 gene was evaluated using methylation-sensitive restriction enzymes, and reversal of the transcriptional block of the p16 gene was determined by Northern blot analysis after treatment with 5-aza-2'-deoxycytidine.
Results:
Homozygous deletions of the p16 and 15 genes from 2 of 9 gastric carcinoma cell lines were found. In contrast, no deletions were detected in 36 primary gastric tumors, and one primary tumor showed rearrangements of the p16 and p15 genes. Two gastric carcinoma cell lines showed a point mutation and an insertional mutation of the p16 gene, respectively; however, no point mutations were noted for the p16 and p15 genes in any of the primary gastric tumors. Constitutive levels of p16 mRNA expression in gastric carcinoma cell lines were quite heterogeneous; four gastric carcinoma cell lines had no detectable p16 mRNA and 6 gastric carcinoma cell lines had negligible expression of p15 mRNA. Of 10 primary gastric tumors, only 1 tumor expressed p16 mRNA. Furthermore, abnormal DNA methylation patterns of the p16 gene were found in 2 gastric carcinoma cell lines through the use of methylation-sensitive restriction enzymes. These cell lines lacked expression of p16 mRNA without deletions of the p16 gene. These transcriptional blocks were reversed by treatment with 5-aza-2'-deoxycytidine.
Conclusions:
Deletions or mutations of the p16 and p15 genes are uncommon in primary gastric carcinomas. However, defective mRNA transcription, sometimes by aberrant DNA methylation, might be one of the pathways of inactivation of the p16 gene that leads to the development of gastric carcinoma.
Insights
Alterations in p16 and p15 genes are rare in gastric cancer. However, abnormal DNA methylation and defective mRNA transcription of the p16 gene may contribute to gastric carcinoma development.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Cyclin-dependent kinase inhibitors (CDKIs), including p16 and p15, are recognized as tumor suppressor genes.
- Alterations in CDKIs are prevalent across various cancer types.
- The specific status of p16 and p15 genes, particularly promoter methylation, in gastric carcinoma remains underexplored.
Purpose of the Study:
- To investigate the alterations, including deletions, mutations, and methylation, of the p16 and p15 genes in gastric carcinoma.
- To assess the expression levels of p16 and p15 mRNA in gastric tumors and cell lines.
- To determine the role of DNA methylation in the inactivation of the p16 gene in gastric carcinoma development.
Main Methods:
- Analysis of p16 and p15 gene alterations (deletion, mutation) in 36 primary gastric tumors and 9 cell lines using Southern blot, Northern blot, and PCR-SSCP.
- Evaluation of p16 gene promoter methylation using methylation-sensitive restriction enzymes.
- Assessment of transcriptional reversal of p16 gene silencing via 5-aza-2'-deoxycytidine treatment.
Main Results:
- Homozygous deletions of p16 and p15 were found in 2 of 9 gastric carcinoma cell lines, but not in primary tumors.
- Mutations in p16 and p15 were rare in primary gastric tumors, though present in cell lines.
- Aberrant DNA methylation of the p16 gene was observed in cell lines, leading to suppressed mRNA expression, which was reversible with 5-aza-2'-deoxycytidine.
Conclusions:
- Deletions and mutations of p16 and p15 genes are infrequent in primary gastric carcinomas.
- Defective mRNA transcription, potentially driven by aberrant DNA methylation, represents a significant mechanism for p16 gene inactivation in gastric carcinoma.
- These findings highlight the importance of epigenetic modifications in the pathogenesis of gastric cancer.