Cyclin A promoter study. (A) CpG island sequence. Human cyclin A promoter CpG island (−131/138) contains 23 CpG sites (bold) and 3 consensus elements (underlined). Cis-acting transcription factors that bind to cyclin A promoter include ATF/CREB, CDE/CHR, and E2F. (B) Methylation status bisulfate genomic DNA sequencing. HUVECs were treated with 50 μM DL-Hcy for 48 hours. Genomic DNA was extracted and modified by bisulfite to convert all unmethylated cytosines to uracils. Bisulfite-modified DNAs were amplified with cyclin A–specific primers, cloned into the TA cloning vector, and sequenced. Unmethylated cytosines are indicated by stalks. Methylated cytosines are indicated by stalks with oval heads. (C) Promoter activities. Plasmids containing cyclin A promoter (−133/+205) with mutations on each consensus element were constructed with the luciferase (Luc) gene in PGL2 vector. HUVECs were transiently transfected with 2 μg PGL2 plasmid DNA by Lipofectin transfection. Cells were treated with 50 μM DL-Hcy in control medium 24 hours after transfection, and harvested 24 hours later. The corrected luciferase activity for each time point was divided by that of control plasmid in cells not treated with Hcy, and is presented as relative luciferase activity. Values represent the mean (± SD) from 3 separate experiments (n = 9). *P < .01 versus control.