Aberrant p21 methylation in acute lymphoblastic leukemia.

(A) A schematic map of the p21 promoter for restriction sites and the position of a primer set. Arrow indicates the location of the transcription initiation site of the p21 gene. Six Sp1-binding sites are located in a GC-rich region within 180 base pairs (bp) upstream of the transcription initiation site of the p21 gene. The −124 and −111 indicate the recognition sites of the restriction enzymeHpaII. Two arrows indicate a primer set for PCR and the size of PCR product, 110bp. (B) Methylation status of the p21 promoter region was detected by means of PCR of the HpaII-digested DNA. The upper band represents the 110-bp amplification product of the p21 gene, and the lower band, 75 bp of the β-actin gene. The optical density curves of both fragments were used to quantify the methylation level of p21 gene. The mean p21/β-actin ratio in normal bone marrow and the SE were used to assess p21 methylation state. Ratios above 3 SEs were considered hypermethylated. Lanes 1-5 show ALL samples. The p21–to–β-actin ratios are shown below the gel, with hypermethylated status indicated by asterisks. (C) Expression of p21 messenger RNA (mRNA) assessed by reverse-transcriptase (RT) PCR by means of primers for p21 and RARα (as control). Lanes 1, 2, and 5: hypermethylated ALL samples showing lack of p21 expression. Lanes 3, 4: normally methylated samples.