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Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
Published on: June 16, 2017
Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia
1Department of Pediatrics, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.
Insights
Inactivation of the p16 and p15 genes through methylation is common in acute leukemia, impacting the RB/p16 tumor suppressive pathway. This study investigated gene methylation in leukemia cell lines and patients, revealing frequent inactivation events crucial for leukemia development.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- The RB/p16 tumor suppressive pathway, involving p16 and p15 as cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, is frequently abrogated in human neoplasms.
- While deletions at the p16/p15 locus are specific to lymphoid malignancies like T-cell acute lymphoblastic leukemia (T-ALL), they are rare in other ALL subsets.
- Investigating alternative inactivation mechanisms like gene methylation is crucial for understanding leukemia pathogenesis.
Purpose of the Study:
- To determine the frequency of p16 and p15 gene inactivation via 5' CpG island methylation in acute leukemia.
- To assess the correlation between gene methylation, protein expression, and the RB/p16 pathway in leukemia.
Main Methods:
- Analysis of 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients.
- Utilized Southern blot, polymerase chain reaction (PCR), and Western blot analyses to detect gene methylation and protein expression.
- Examined RB protein expression and alterations in cyclin D1 and CDK4.
Main Results:
- p16 methylation was found in 50% of ALL cell lines, 50% of AML cell lines (without homozygous deletion), and 38% of AML patients, correlating with loss of p16 protein expression.
- p15 methylation occurred in 34% of ALL cell lines, 60% of AML cell lines (without homozygous deletion), and 52% of AML patients.
- No RB protein expression was observed in 4 of 16 leukemia cell lines, and p16 inactivation was present in all cell lines with RB expression. No cyclin D1 amplification or rearrangement was detected.
Conclusions:
- Methylation-induced inactivation of p16 and p15 genes is a frequent event in acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML).
- These genetic events play a significant role in the RB/p16 pathway's disruption, contributing to the pathogenesis of acute leukemia.
- Methylation represents a key mechanism for p16/p15 inactivation in leukemias, complementing information from deletion and mutation studies.
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