Cytosine methylation in the promoter region of the INK4 cyclin-dependent kinase inhibitor (CDKI) gene p!5INK4B is associated with loss of detectable corresponding mRNA in human acute myeloid leukemia (AML). To determine if members of the KIP family of CDKIs are also associated with CpG promoter methylation, we compared p!5INK4B patterns of promoter methylation and mRNA levels with KIP family members p27KIPl and p21 WAF1 in 35 consecutive AML cases. Methylation was assessed by PCR amplification of promoter fragments from bisulfite treated genomic DNA, resolution of differentially methylated sequences by temporal temperature gradient electrophoresis, followed by cloning and sequencing. Thirty, 55 and 35 CpG sites were analyzed within the promoters and 5' untranslated regions of p!5INK4B, p27KIPl and p21WAFl, respectively. Partially methylated sequences were present in all three promoters in all AML cases with the greatest degree and range of methylation observed in the p!5INK4B promoter. The percentage of methylated CpG sites ranged from 0 to 100% in clones of the p 15INK4B promoter compared with 0 to 63.4% for p27KIPl and 0 to 14.3% for p21WAFl. The p!5INK4B promoter harbored clones containing fully methylated sequences in 30/32 (93.8%) AML cases, while the p27KIP 1 and p21 WAF1 promoters lacked clones containing fully methylated sequences in all cases. By RT-PCR, 26/35 (74.3%) AML cases lacked detectable p!5INK4B mRNA, whereas only 3/35 (8.6%) lacked p27K!Pl mRNA and 0/35 (0%) lacked detectable p21WAF1 mRNA. Our data indicates that similar to p 15INK4B, heterogeneous methylation of multiple CpG sites occurs within the p27KIPl promoter in primary cases of human AML. However, unlike p!5INK4B, methylation of the p27KIPl promoter is not associated with mRNA loss, despite the existence of clones containing over 60% methylated CpG sites. The absence of fully methylated sequences within the p27KIPl promoter may explain, in pan, the differing effects of promoter methylation on p 15INK4B and p27KIP 1 mRNA expression. The p21WAFl promoter appears to be methylated only to a minor extent in human AML.
|Issue number||11 PART II|
|Publication status||Published - 2000 Dec 1|
ASJC Scopus subject areas
- Cell Biology