Figure 4
Figure 4. Effects of 5-azacytidine and decitabine on the 5′methylation status of FAS and FAS mRNA expression levels in neoplastic MCs. (A) HMC-1 cells were exposed to control medium (untreated), 5-azacytidine (5μM) or decitabine (5μM) for 96 hours. Then, the 5′methylation status of p15, p16, p21, and FAS was determined by MSP. EpiTect-methylated control DNA was used as positive-control. Vertical lines have been inserted to indicate a repositioned gel lane. (B) 5′methylation status of p15, p16, p21, and FAS in normal BM cells in 2 donors (#1 and #2) determined by MSP. To verify efficient sodium bisulfite conversion, we also performed unmethylated specific PCR (USP) for p15, p16, p21, and FAS. EpiTect-methylated and unmethylated control DNA were used as positive-control. (C) BGS of a part of the FAS CGI was performed as previously described using HMC-1.1 cells, HMC-1.2 cells, and normal BM cells. Black squares indicate methylated cytosines at CpG sites, and white squares represent unmethylated cytosines at CpG sites. Whereas in HMC-1.1 cells, 80% of all cytosines at CpG sites analyzed were methylated, 15% of all cytosines at CpG sites analyzed were methylated in HMC-1.2 cells. No methylation was detected in normal BM cells. The percentage of methylation and the significance by Fisher exact test (A) and χ2 test (B) are shown. The bottom panel shows representative chromatograms from BGS in HMC-1.1 cells, HMC-1.2 cells, and normal BM cells. Sites for methylation are underlined. Asterisks indicate cytosines that were converted to thymine. (D) HMC-1.1 and HMC-1.2 cells were incubated in control medium, 5-azacytidine (5μM) or decitabine (5μM) at 37°C for 48 hours (gray bars) or 96 hours (black bars). Thereafter, FAS mRNA expression was analyzed by qPCR. GAPDH served as a reference gene. Results show the fold-increase of mRNA expression and represent the mean ± SD of 3 independent experiments (*P < .05 compared with control). (E) HMC-1 cells were incubated with control medium, 5-azacytidine, or decitabine (each 5μM) in the absence or presence of FAS-ligand (1 ng/mL) for 96 hours. Then, annexin V/PI staining was performed. Results show the percentage of annexin V/PI+ cells. (F) HMC-1 cells were incubated in control medium, 5-azacytidine, or decitabine (each 5μM) in the absence (black bars) or presence (gray bars) of FAS-ligand (1 ng/mL) for 96 hours. Then, the numbers (percentage) of active caspase-3–positive cells were assessed by flow cytometry. Results show the mean ± SD of 3 independent experiments.

Effects of 5-azacytidine and decitabine on the 5′methylation status of FAS and FAS mRNA expression levels in neoplastic MCs. (A) HMC-1 cells were exposed to control medium (untreated), 5-azacytidine (5μM) or decitabine (5μM) for 96 hours. Then, the 5′methylation status of p15, p16, p21, and FAS was determined by MSP. EpiTect-methylated control DNA was used as positive-control. Vertical lines have been inserted to indicate a repositioned gel lane. (B) 5′methylation status of p15, p16, p21, and FAS in normal BM cells in 2 donors (#1 and #2) determined by MSP. To verify efficient sodium bisulfite conversion, we also performed unmethylated specific PCR (USP) for p15, p16, p21, and FAS. EpiTect-methylated and unmethylated control DNA were used as positive-control. (C) BGS of a part of the FAS CGI was performed as previously described using HMC-1.1 cells, HMC-1.2 cells, and normal BM cells. Black squares indicate methylated cytosines at CpG sites, and white squares represent unmethylated cytosines at CpG sites. Whereas in HMC-1.1 cells, 80% of all cytosines at CpG sites analyzed were methylated, 15% of all cytosines at CpG sites analyzed were methylated in HMC-1.2 cells. No methylation was detected in normal BM cells. The percentage of methylation and the significance by Fisher exact test (A) and χ2 test (B) are shown. The bottom panel shows representative chromatograms from BGS in HMC-1.1 cells, HMC-1.2 cells, and normal BM cells. Sites for methylation are underlined. Asterisks indicate cytosines that were converted to thymine. (D) HMC-1.1 and HMC-1.2 cells were incubated in control medium, 5-azacytidine (5μM) or decitabine (5μM) at 37°C for 48 hours (gray bars) or 96 hours (black bars). Thereafter, FAS mRNA expression was analyzed by qPCR. GAPDH served as a reference gene. Results show the fold-increase of mRNA expression and represent the mean ± SD of 3 independent experiments (*P < .05 compared with control). (E) HMC-1 cells were incubated with control medium, 5-azacytidine, or decitabine (each 5μM) in the absence or presence of FAS-ligand (1 ng/mL) for 96 hours. Then, annexin V/PI staining was performed. Results show the percentage of annexin V/PI+ cells. (F) HMC-1 cells were incubated in control medium, 5-azacytidine, or decitabine (each 5μM) in the absence (black bars) or presence (gray bars) of FAS-ligand (1 ng/mL) for 96 hours. Then, the numbers (percentage) of active caspase-3–positive cells were assessed by flow cytometry. Results show the mean ± SD of 3 independent experiments.

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