Figure 2

Global hypermethylation in MCF-7/Adr cells. a) 5-mC content was evaluated by capillary electrophoresis. Percentage of 5-mC was 3.6% in MCF-7, while this increased to 5.6% in MCF-7/Adr cells; b) cytosine-extension assay based on use of methylation-sensitive restriction endonuclease that leaves a 5' guanine overhang after DNA cleavage with subsequent single-nucleotide extension with radiolabeled [(3)H]dCTP shows a 40% decrease in incorporation of radiolabeled [(3)H]dCTP by the DNA of HpaII-digested MCF-7/Adr cells, indicative of hypermethylation, and c), a polyclonal antibody against 5-methylcytosine was incubated with the digested DNA spotted in a nitrocellulose membrane. Spot intensity is 30% higher as evaluated by densitometric analysis in the DNA of MCF-7/Adr cells, also indicative of DNA hypermethylation.