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Published on: February 24, 2015
Methylation of the p16INK4A gene in multiple myeloma
1Department of Medicine, UCLA School of Medicine, Cedars-Sinai Research Institute, Los Angeles, California 90048, USA.
Abstract:
The p16INK4A (p16) binds to both cyclin D-CDK4 and cyclin D-CDK6 and inhibits the progression of the cell cycle from G1 to S phase. Loss of expression of this protein can occur by several mechanisms including structural alterations. Recent studies have suggested that the loss of expression of p16 can occur by hypermethylation of the gene. The methylation status of the p16 gene in multiple myeloma was examined in three myeloma cell lines (U266, RPMI8226 and IM9) and 16 primary myeloma samples using methylation-specific polymerase chain reaction (MSP). The U266 and RPMI8226 cell lines contained a completely methylated p16 gene and the IM9 line had a partially methylated p16 gene. Identical results were obtained by another polymerase chain reaction (PCR)-based methylation assay system as well as Southern blotting after using a methylation-sensitive restriction enzyme. The U266 cell line expressed no p16, and the IM9 had weak expression as determined by reverse transcript (RT-)PCR. The U266 cells began to express, and IM9 increased the accumulation of, the p16 RNA after treatment with the demethylating agent 5'-aza-2-deoxycytidine (10(-6)-10(-5) M). This suggested that the levels of methylation of the p16 gene detected by the MSP technique correlated with the regulation of transcription of this gene. Examination of the primary myeloma samples showed that eight of 16 (50%) contained a methylated p16 gene. We have previously found that alterations of the p16 gene, such as deletions and point mutations, are rare in primary multiple myeloma; none of the 16 samples included in this study had p16 gene alterations. Our results suggest that methylation of the p16 gene may contribute to the development and/or progression of multiple myeloma.
Insights
Methylation of the p16INK4A (p16) gene is frequent in multiple myeloma, potentially driving disease development. This epigenetic silencing correlates with reduced p16 expression, unlike genetic alterations.
Area of Science:
- Oncology
- Epigenetics
- Molecular Biology
Background:
- The p16INK4A (p16) protein regulates cell cycle progression from G1 to S phase by inhibiting cyclin D-CDK4/6 complexes.
- Loss of p16 expression, a tumor suppressor, can occur through genetic alterations or epigenetic silencing, such as gene hypermethylation.
- The role of p16 gene methylation in the pathogenesis of multiple myeloma requires further investigation.
Purpose of the Study:
- To investigate the methylation status of the p16 gene in multiple myeloma cell lines and primary patient samples.
- To correlate p16 gene methylation levels with p16 expression and explore the impact of demethylating agents.
Main Methods:
- Methylation-specific polymerase chain reaction (MSP) was employed to assess p16 gene methylation in three myeloma cell lines (U266, RPMI8226, IM9) and 16 primary myeloma samples.
- Confirmatory methylation analysis was performed using an alternative PCR-based system and Southern blotting with methylation-sensitive restriction enzymes.
- Reverse transcriptase PCR (RT-PCR) was used to determine p16 gene expression levels, and demethylating agent treatment was applied to assess transcriptional regulation.
Main Results:
- Complete methylation of the p16 gene was observed in U266 and RPMI8226 cell lines, and partial methylation in the IM9 cell line.
- p16 expression was absent in U266 and weak in IM9 cells; demethylating agent treatment restored or increased p16 RNA levels, indicating methylation-dependent transcriptional silencing.
- Fifty percent (8 of 16) of primary multiple myeloma samples exhibited p16 gene methylation, while genetic alterations like deletions or point mutations were rare.
Conclusions:
- Epigenetic silencing of the p16 gene through methylation is a common event in multiple myeloma.
- p16 gene methylation correlates with reduced p16 expression and may play a significant role in the development and progression of multiple myeloma.
- Targeting epigenetic modifications could be a potential therapeutic strategy for multiple myeloma.
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