Figure 1.
AZA and DAC differentially affect cell viability in AML cell lines.
Cell viability of AML cell lines, KG-1a, THP-1, OCI-AML3, and HL-60, was assessed after 72 hours of treatment with AZA (•) or DAC (□) (0–50 µM) using the CellTiter-Glo assay. Standard deviation was determined from 2 or 3 independent experiments, including triplicate wells per experiment. AML = acute myeloid leukemia; AZA = azacitidine; DAC = decitabine.
Table 1.
AZA and DAC potencies on acute myeloid leukemia cell viability.
Figure 2.
AZA incorporates into RNA and DNA of KG-1a cells.
KG-1a cells were treated with 0.3 µM radiolabeled AZA ([14C]-AZA) for 24 hours. The amount of AZA incorporated into total nucleic acid, DNA, and RNA was quantified as described previously. Standard error of the mean was determined from 3 independent experiments, including triplicate wells per experiment. AZA = azacitidine.
Figure 3.
AZA inhibits protein synthesis in KG-1a and THP-1 cells.
Cells were treated daily with AZA or DAC (0–5 µM) for 24 or 48 hours prior to metabolic labeling with 35S-methionine and 35S-cysteine. Protein synthesis was quantified as described previously. AZA = azacitidine; DAC = decitabine.
Figure 4.
AZA and DAC cause DNMT1 depletion and induction of DNA damage in KG-1a and THP-1 cells.
Cells were treated daily with AZA or DAC (0–3 µM in KG-1a; 0–10 µM in THP-1) for 48 and 72 hours. Protein lysates were analyzed by Western analysis for DNMT1 and phospho-H2AX (Ser 139) proteins. α-Tubulin is shown as a protein loading control. AZA = azacitidine; DAC = decitabine; DNMT = DNA methyltransferase.
Figure 5.
AZA and DAC reduce DNA methylation in KG-1a and THP-1 cells.
Cells were treated daily with AZA (•) or DAC (□) (0–3 µM) for 48 hours. DNA methylation was measured using (A) pyrosequencing of LINE-1 DNA elements in bisulfite-converted DNA and (B) Illumina GoldenGate platform. AZA = azacitidine; DAC = decitabine.
Figure 6.
Effects of AZA and DAC on cell cycle and apoptosis in KG-1a cells.
KG-1a cells were treated daily with AZA or DAC (0–3 µM) for 48 hours. (A) Cell cycle effects of AZA and DAC. Cells were stained with NIM-DAPI and quantification by flow cytometry for percentage of cells in sub-G1, G0/G1, S, and G2-M phases (normalized to 100%). (B) AZA and DAC induce apoptosis in KG-1a cells. Apoptosis was detected with flow cytometry by positive staining for Annexin V (early apoptosis) and 7-AAD (late apoptosis). (C) Protein lysates were analyzed by Western analysis for detection of PARP cleavage. α-Tubulin is shown as a protein loading control. AZA = azacitidine; DAC = decitabine.
Figure 7.
AZA and DAC regulate different genes in KG-1a cells.
Venn diagrams reveal the number of genes that are distinctly and commonly regulated by daily treatment with AZA (1 µM) or DAC (0.3 µM or 1 µM) in KG-1a cells at 24 and 48 hours. AZA = azacitidine; DAC = decitabine.
Table 2.
Number of genes regulated by AZA and DAC in KG-1a cells.
Table 3.
Gene biogroups significantly regulated by AZA or DAC in KG-1a cells.