Cyclin D1 promoter DNA was unmethylated in normal human B and T cells. Southern blot analysis with the cyclin D1 promoter and the MTC probes and bisulfite sequencing analysis in the cyclin D1 promoter region. (A) EBV-transformed human B cells. (B) Normal human CD19⁺ B cells. (C) Normal human CD4⁺ T cells. (D) Patient samples with cyclin D1⁺ B-cell malignancies. For normal B and T cells, the cyclin D1 promoter was demethylated even though the gene was not expressed (Figure 1B). However, the MTC region was methylated. In 2 MCL bone marrow samples from patients (Pt 2 and Pt 3) and in a peripheral blood sample from a patient with lymphocytosis and t(11;14) B-cell malignancy (Pt 1), both the cyclin D1 promoter region (Pts 1-3) and the MTC region (Pt 1) were unmethylated in purified B cells. The methylation differences in the MTC region were confirmed by bisulfite sequencing (not shown), although the number of CpG dinucleotides in the MTC is small.