Fig. 1.

Disulfiram inhibits DNMT1 in vitro and results in reduction of ^5meC content in prostate cancer cells. A: DNMT1enzyme activity assays were performed by incubating recombinant His6-DNMT1 with hemimethylated oligonucleotide substrates and S-adenosyl-L-[methyl-³H] methionine as a methyl group donor in the presence of solvent control alone or DSF at increasing concentrations. Relative enzymatic activity is expressed as percentages of solvent control. B: DNMT1is expressed at high levels in prostate cancer cell lines but not in normal PrECs. Normal PrEC and prostate cancer cell lines (CWR22Rv1, PC3, C4-2B, DU145) were lysed and lysates were subjected to SDS^PAGE separation. Proteins were blotted onto PVDF membranes and were incubated with anti-DNMT1 and anti-Actin-specific antibodies. Desitometric analyses show DNMT1 band intensities normalized to Actin. C: LC-MS/MS analysis was used to determine ^5meC as a fraction of total cytosine content in CWR22Rv1 and PC3 cells treated with DSF for indicated time points. Normal white blood cell (WBC) genomic DNA in which all CpG dinucleotides were methylated to completion with the M.SssI methyltransferase in vitro (WBC-SssI) was used as positive control. *P <0.05.