Figure 2

Global demethylation and histone acetylation restores SFRP2 expression. (A) MSP of four malignant cell lines was performed with DNA from either untreated cells, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. In three cell lines (BT20, MCF7, T47D) a promoter demethylating effect could be visually detected, since signals indicative of unmethylated SFRP2 promoter arise (BT20, MCF7) or become enhanced (T47D) after the combined treatment. In T47D, DAC alone had no detectable demethylating effect on the SFRP2 promoter. (B) Expression of SFRP2 mRNA before treatment, or after treatment with 1 μM DAC, or after treatment with 300 nM TSA, or after a combined treatment applying both drugs. Treatment with DAC alone was not able to induce SFRP2 expression in all cell lines, in contrast to TSA which induced expression in two out of four cell lines (SKBR3 and T47D) previously showing partial SFRP2 methylation. However, only combined promoter demethylation and histone reacetylation leads to strong induction of SFRP2 mRNA expression in all cell lines. GAPDH served as cDNA loading control. (C) Suppression of Cyclin D1 mRNA expression after global DNA demethylation of breast cancer cell lines as determined by realtime PCR. Untreated tumor cells (black bars) and cells treated with DAC/TSA (grey bars) show significantly different expression levels of Cyclin D1 mRNA (P = 0.029, two-sided Mann-Whitney U-test). Expression level of each sample is normalized to its GAPDH expression and related to untreated BT20 cells (set to 1).