Progressive De Novo DNA Methylation at the Pa Promotor of the Abl Genein the Bcr-Abl Locus during the Course of Chronic Myelogenous Leukemia

Yang, Sung Eun; Chi, Hyun Sook

Abstract

BACKGROUND: In most cases of chronic myelogenous leukemia (CML), one of the two abl promotors (Pa) is nested within the bcr-abl hybrid gene and should be able to transcribe the 6-kb normal abl mRNA from the Philadelphia chromosome. However, 6-kb transcript is present only in CML cell lines containing a normal abl allele. Since the nested Pa in the bcr-abl hybrid gene is contained within a CpG island, de novo methylation of Pa CpG islands may cause silencing of the c-abl gene. In this study, using a polymerase chain reaction (PCR) and methylation-sensitive restriction enzymes, we investigated the methylation status of the nested Pa CpG islands in the bcr-abl hybrid gene at two stages of CML and the usefulness of it as a disease progression marker.
METHODS
Genomic DNA was extracted from the preserved bone marrow smear slides of ten CML patients, at chronic phase and blast crisis respectively. K-562 cell line and normal peripheral lymphocytes as a negative control were also used. The extracted genomic DNA was digested with methylation-sensitive restriction enzymes and methylation-insensitive restriction enzymes, respectively, followed by PCR amplification for Pa promotor and electrophoresed for detection of 171 bp PCR products.
RESULTS
The patients were transformed from chronic phase to blast crisis during 11-60 months after initial diagnosis. The patients displayed the following three types of methylation patterns. A, no methylation; B, partial methylation; C, complete methylation. All cases of chronic phase showed pattern A, but three cases of blast crisis were pattern B and seven cases, pattern C.
CONCLUSIONS
De novo DNA Methylation at the Pa promotor of the abl gene in the bcr-abl hybrid gene is a distinct and common molecular event in blast crisis of CML. Since the process of Pa CpG methylation is of a progressive nature, Pa methylation pattern may be used as a molecular marker for progression of CML to blast crisis.