DAP-kinase CpG island methylation in acute myeloid leukemia: methodology versus biology?

Acute myeloid leukemia (AML) is a clonal disorder evolving from myeloid progenitor cells on the background of a number of genetic changes including balanced translocations1,2 and alterations of tumor suppressor genes and proto-oncogenes.3Recently, transcriptional silencing of tumor suppressor genes by hypermethylation of promoter CpG islands has emerged as an event that may contribute to the leukemic phenotype. Among the genes found to be hypermethylated at significant rates in AML arep15^INK4B,4-6 the estrogen receptor,7 and HIC1.8 By this letter we would like to draw attention to methodological features that may cause confusion in the interpretation of data on promoter methylation.

In a recent issue of Blood, Katzenellenbogen et al9reported that hypermethylation of the death-associated protein kinase (DAP-kinase) is a common alteration in B-cell malignancies. DAP-kinase is a 160-kd cytoskeleton-associated protein with serine/threonine kinase activity. It functions as a positive mediator of interferon-γ induced cell death10 and is furthermore involved in FAS and TNF-α induced apoptosis.11 Using a methylation-specific polymerase chain reaction (MSP) assay12Katzenellenbogen et al found that 100% (9 of 9) of samples from Burkitt's lymphoma and 84% (21 of 25) from other B-cell lymphoma patients were methylated in the DAP-kinase 5′ CpG island. In contrast, no methylation was found in T-cell lymphoma or T-cell acute lymphoblastic leukemia samples. In AML, 3.8% (1 of 26) of pediatric samples and 33% (2 of 6) of adult samples were found to contain methylated DAP-kinase alleles.

We recently developed a novel method, “bisulfite-DGGE,” that detects aberrant methylation and provides a detailed display of the composite pattern of clonotypic epigenotypes within a sample of DNA.6 This method combines bisulfite treatment of genomic DNA with nondiscriminatory PCR amplification of methylated and unmethylated sequences, followed by resolution of alleles with varying methylation density by denaturing gradient gel electrophoresis (DGGE). By using this method, we demonstrated a highly heterogeneous pattern of p15^INK4Bmethylation in AML samples.6 

Prompted by the above mentioned report,9 we analyzed leukemic blasts from AML patients and leukemic cell lines for methylation in the DAP-kinase CpG island using both MSP and bisulfite-DGGE. MSP was performed with primers identical to those described by Katzenellenbogen et al,9 while for bisulfite-DGGE, we designed a pair of primers to amplify a 103-bp region of the DAP-kinase CpG island, which contains 7 CpG sites and covers essentially the region examined by Katzenellenbogen et al.9 

In concordance with the previous report,9 we found, by both MSP and bisulfite-DGGE analysis, that the Raji cell line is completely methylated in the DAP-kinase CpG island, and that the HL-60 cell line is partially methylated (Figure). MSP analysis of clinical AML samples revealed methylated alleles of the DAP-kinase in 42% (19 of 45) of de novo adult AML cases and in none of four de novo pediatric AML cases (Figure). Direct sequence analysis of the PCR products showed that all cytosines at CpG sites had remained as cytosine during bisulfite treatment and all cytosines at non-CpG sites were converted to thymine (data not shown), excluding the possibility that successful amplification could be due to unspecific primer annealing or incomplete bisulfite conversion. Bisulfite-DGGE was performed on the same 49 AML cases and Figure 1B shows bisulfite-DGGE experiments of the same samples as those analyzed by MSP in Figure 1A. For all AML samples, the vast majority of amplified DAP-kinase alleles migrated to a position in the gel corresponding to unmethylated DNA (Figure), and a band pattern consistent with some degree of DAP-kinase promoter methylation could be demonstrated in only 1 case of adult AML (data not shown). For comparison, the 10 AML samples represented in the Figure showed extensive methylation of the p15^INK4B CpG island by bisulfite-DGGE analysis (Figure).

The direct comparison between the MSP and DGGE assays performed in this study prompts us to suggest that silencing of DAP-kinase by methylation may not be a biologically significant event in AML. This is evidenced by the fact that by MSP analysis, 42% of adult AML cases showed indication of DAP-kinase CpG island methylation, whereas bisulfite-DGGE of the same samples revealed that only an insignificant fraction of leukemic blasts, or possibly even a different cell type, contained methylated DAP-kinase alleles. These contrasting results may be reconciled by data in a recent study where we found that a small fraction of normal lymphocytes are methylated in the promoter region ofp15^INK4B.6 The presence of rare nonmalignant methylated cells in clinical leukemia samples implies that highly sensitive methods for detection of methylation, such as the MSP, should be employed with caution and preferably in combination with other methods in order to obtain information on both the fraction and the extent of methylated alleles. In this setting, and when seen in conjunction with accepted clinical correlates, promoter methylation could, however, well turn out to be of significance in stratifying patients with hematologic diseases.