Interdependence of histone hypoacetylation and DNA hypermethylation at the Prdx2 promoter in AML. (A) Expression of PRDX members in different leukemic cell lines was analyzed by Western blot analysis, showing cell lines with high (KCL22, KG1, Kasumi) and low (U937, HL60, NB4) PRDX2 protein expression. (Right) Induction of ROSs by H2O2 treatment in different leukemic cell lines measured by CM-H2DCFDA dye. (B) H3Ac at the PRDX2 promoter in leukemic cell lines was analyzed by ChIP and real-time PCR with the use of anti-H3Ac Ab. Enrichments for the H3 acetylation Ab are calculated against IgG control with the use of the ddCt method. Error bars represent SD calculated from triplicate quantitative PCR reactions. The results were verified in ≥ 2 independent biologic experiments. (C) H3Ac was increased at the PRDX2 promoter in U937 cells after 5-azacytidine treatment as analyzed by ChIP. U937 cells were seeded at a density of 5 × 105/mL with 5-azacytidine (Sigma-Aldrich) at concentration of 100nM. Cells were harvested after 2, 4, and 6 days of treatment for ChIP, RNA, or DNA preparation. Enrichments for the H3 acetylation Ab are calculated against IgG control with the use of the ddCt method. Error bars represent SD calculated from triplicate quantitative PCR reactions. The results were verified in ≥ 2 independent biologic experiments. (D) PRDX2 mRNA expression was induced after 5-azacytidine treatment in U937 cells as analyzed by real-time RT-PCR. Error bars represent SD calculated from 3 independent biologic experiments. Primer sequences are given in the supplemental information for methods. (E) PRDX2 promoter DNA methylation was decreased in the U937 cell line after 5-azacytidine treatment as analyzed by MSP. Nonmethylated promoter (USP) and methylated promoter regions (MSP) were analyzed by PCR. (Inset) Densitometry of the MSP gels indicates a decrease in methylation levels at the PRDX2 promoter after 5-azacytidine treatment in U937 cells. The figure represents the mean ± SD of densitometric values obtained from 3 independent gels. (F) H3Ac at the PRDX2 promoter in U937 cells after treatment with azacytidine in combination with TSA or TSA alone analyzed by ChIP. U937 cells were seeded at a density of 5 × 105/mL with 5-azacytidine (Sigma-Aldrich) at a concentration of 100nM for 3 days followed by addition of TSA (1μM) for 1 hour. Enrichments for the H3 acetylation Ab or IgG were calculated against input with the use of the ddCt method. Error bars represent SD calculated from duplicate quantitative PCR reactions. (G) PRDX2 mRNA expression was induced after 5-azacytidine treatment or combination of 5-azacytidine and TSA in U937 cells as analyzed by real-time RT-PCR. Relative expression was calculated against GAPDH by ddCt method. Error bars represent SD calculated from 2 independent biologic experiments. Primer sequences are given in the supplemental information for methods.
