Increasing methylation of the CDKN2A gene is associated with the progression of adult T-Cell leukemia

In this study, we examined the methylation status of the CDKN2A gene in pat ients with different forms of adult T-cell leukemia (ATL) using Southern bl ot analysis, methylation-specific PCR (MSPCR), and nucleotide sequencing. W e found that the CDKN2A gene was more frequently methylated in fresh tumor cells isolated from patients with acute ATL (47%) or lymphoma-type ATL (73% ) than in those with less malignant chronic (17%) and smoldering (17%) ATL. In addition, deletions of the CDKN2A gene were found in 24% of acute ATL p atients; thus, abnormalities of the CDKN2A gene totaled 71% in acute ATL pa tients. In contrast, no CDKN2A gene methylation was found in asymptomatic c arriers or uninfected individuals. Methylation of the p15 gene was not foun d in any samples from 36 ATL patients. Direct sequencing of the CDKN2A gene after sodium bisulfite treatment of genomic DNA revealed that the methylat ion of CpG sites had occurred in 24 of 32 ATL cases (75%) including chronic and smoldering ATL, even when MSPCR and the Southern blot had failed to de tect CDKN2A gene methylation. Among fresh ATL samples with methylation, met hylation was detected in the promoter region and exon in 17 of 24 cases, an d methylation in the exon without promoter region was detected in 17 of 24 cases. In one case, the pattern of methylation proved to be different betwe en peripheral blood cells and lymph node cells, suggesting the presence of multiple subclones with regard to methylation patterns, despite the same HT LV-I integration site. Quantitative PCR showed a marked decrease in CDKN2A mRNA expression in the cells with a methylated CDKN2A gene, especially if t he promoter region was methylated. These findings suggest that CpG methylat ion decreases CDKN2A expression and represents a critical factor in the dis ease progression of ATL.