FIG. 2.

Promoter methylation of CDKN1A is involved in suppression of CDKN1A expression in ATLL cells. (A) Semiquantitative RT-PCR of CDKN1A expression was performed with mRNA isolated from PBLs from two healthy volunteers and leukemia cells from two acute-type ATLL patients (Pt 1 and 2) after treatment with 10 μM 5-aza-dC for 72 h or with 1.2 μM TSA for 24 h. The HUT102 cell line was used as a control. (B) Quantitative RT-PCR of CDKN1A expression was performed with mRNA isolated from various T-leukemia cell lines treated with 10 μM 5-aza-dC for 72 h, with 1.2 μM TSA for 24 h, or with 1.2 μM TSA for 24 h followed by 10 μM 5-aza-dC for 48 h. CDKN1A mRNA expression levels were normalized to β-actin mRNA expression and are expressed relative to the mRNA level in the normal CD4⁺ T lymphocytes. Proviral DNA copy numbers for HTLV-1 were measured in each cell line. Statistical analysis was done by a Student's t test. (C and D) MSP analysis of the CDKN1A gene promoter. DNAs from various T-leukemia cell lines (C) and primary ATLL cells (D) were treated with bisulfite and subjected to MSP. A product in the U gel indicates that the CDKN1A gene is unmethylated; a product in the M gel indicates that the gene is methylated. CD4⁺ T lymphocytes of healthy volunteers were used as a control.