No abstract

The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.

Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation-specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non-resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR-ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non-resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.

DNA Methylation, 5meC, is an epigenetic modification that acts as an important regulator of genomic stability and gene expressivity. Genome-wide changes in methylation have been associated with lineage-specific changes in gene expression profiles during development and in some cell-based pathologies, including oncogenesis. Cost-effective and rapid platforms for the detection of changes in the global levels of methylation are of value for the investigation of the processes that regulate methylation. Flow cytometry allows rapid and quantitative analysis of epitopes within a large number of cells. We have recently optimised the conditions required for valid detection of 5meC by immunofluorescence microscopy. These studies showed that immunological detection of 5meC requires the sequential denaturation of chromatin by a brief period of acidification followed by a partial tryptic digestion step. We have assessed the reliability of flow cytometry for the detection of changes in 5meC when coupled with this optimised epitope retrieval strategy. This study provides support for the use of high throughput screening of 5meC by flow cytometry for the analysis of the epigenetic regulation of important cell transitions.