Homocysteine inhibits endothelial cell growth via DNA hypomethylation of the cyclin Agene by Md S. Jamaluddin, Irene Chen, Fan Yang, Xiaohua Jiang, Michael Jan, Xiaoming Liu, Andrew I. Schafer, William Durante, Xiaofeng Yang, and Hong Wang Blood Volume 110(10):3648-3655 November 15, 2007 ©2007 by American Society of Hematology

DNA synthesis and mRNA levels in HUVECs DNA synthesis and mRNA levels in HUVECs. HUVECs were treated with 50 μM DL-Hcy and 1 mM AZC in control medium for 48 hours, or 300 nM TSA and 5 mM NaBr for 24 hours. DNA synthesis and mRNA levels in HUVECs. HUVECs were treated with 50 μM DL-Hcy and 1 mM AZC in control medium for 48 hours, or 300 nM TSA and 5 mM NaBr for 24 hours. (A) Thymidine uptake. Cells were metabolically labeled with 1 μCi (0.037 MBq)/mL [3H]-thymidine during the last 3 hours. Values represent mean (± SD) from 3 separate experiments with triplicates (n = 9). *P < .01 versus non-Hcy and nonviral control. #P < .01 versus Hcy-treated and non–viral-transduced group. (B) Northern blot analysis. Total cellular RNA was used for Northern analysis (10 μg). The blot was hybridized with the probes of human cyclins A and D1, and subsequently with 18S RNA. Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology

Promoter analysis. Promoter analysis. (A) CpG island search. One CpG island is identified in the core promoter region of the cyclin Agene, and 2 in the far 5′-flank region of cyclin D1 gene by computational analysis. (B) Cyclin A promoter activity. Plasmids containing various lengths of the 5′-flanking region of the cyclin A gene were constructed with the luciferase (Luc) gene in PGL2 vector. HUVECs were transiently transfected with 2 μg plasmid DNA by Lipofectin transfection, and harvested 48 hours after transfection. For each construct, 0.5 μg plasmid pCMV-βGAL was cotransfected to correct for differences in the transfection efficiency. The corrected luciferase activity was then divided by that of the cyclin A −266/+205 plasmid and is presented as relative luciferase activity. Values represent mean (± SD) from 3 separate experiments with triplicates (n = 9). Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology

Cyclin A promoter study. 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. Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology

DNMT activities and protein levels. DNMT activities and protein levels. HUVECs were treated with 50 μM DL-Hcy or 1 mM AZC in control medium for 48 hours. Nuclear extracts (20 μg) were prepared and incubated with hemimethylated double-stranded DNA for DNMT1 activity (A) or with unmethylated double-stranded DNA for DNMT3 activity (B) in the presence of S-adenosyl-L-[methyl-3H] methionine. DNMT activities were determined by the radioactivity level of DNA substrates. Values represent mean (± SD) from 3 separate experiments with triplicated samples (n = 9). *P < .01 versus control. Protein levels of DNMT1 and DNMT3 were examined by Western blotting with antibodies against DNMT1 or DNMT3, respectively, and reblotted with β-actin antibody. Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology

The effect of adenovirus-transduced DNMT genes on DNA synthesis and cyclin A mRNA levels in Hcy-treated HUVECs. HUVECs were infected with control adenovirus (Ad-CT) (A), adenovirus expressing DNMT1 (Adv-DNMT1) (B,D), and adenovirus expressing DNMT3 (Adv-DNM... The effect of adenovirus-transduced DNMT genes on DNA synthesis and cyclin A mRNA levels in Hcy-treated HUVECs. HUVECs were infected with control adenovirus (Ad-CT) (A), adenovirus expressing DNMT1 (Adv-DNMT1) (B,D), and adenovirus expressing DNMT3 (Adv-DNMT3) (C) at the indicated MOI for 24 hours, and then treated with 50 μM DL-Hcy in control medium for another 24 hours. (A-C) Thymidine uptake. Cells were metabolically labeled with 1 μCi (0.037 MBq)/mL [3H]-thymidine during the last 3 hours. [3H]-thymidine incorporation was measured in a liquid scintillation counter. Values represent mean (± SD) from 3 separate experiments with triplicates (n = 9). *P < .01 versus non-Hcy and nonviral control. #P < .01 versus Hcy-treated and non–viral-transduced group. Protein levels of DNMT1 and DNMT3 were examined by Western blotting with antibodies against DNMT1 or DNMT3, and reblotted with β-actin antibody. (D) mRNA levels: HUVECs were infected with Adv-DNMT1 for 24 hours and then treated with DL-Hcy in control medium for another 48 hours. Total cellular RNA (10 μg) was extracted and used for Northern analysis. The blot was hybridized with the probes of human cyclin A and D1, and subsequently with 18S RNA. Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology

Chromatin remodeling (CHIP assay). Chromatin remodeling (CHIP assay). HUVECs and HASMCs were treated with 50 μM DL-Hcy and 1 mM AZC in control medium for 48 hours, or 300 nM TSA and 5 mM NaBr for 24 hours. Nuclear extracts were immunoprecipitated with antibody against MeCP2 (A,B), and AcH3 or AcH4 (C). PCR was performed using primers specific for cyclin A promoter from position −267 to 37. Input DNA was amplified to evaluate the total amount of DNA applied to the immunoprecipitation. Md S. Jamaluddin et al. Blood 2007;110:3648-3655 ©2007 by American Society of Hematology