Figure 6.
Epigenetic regulation and relevant leukemic oncogenes repress GADD45g expression in AML, and NF-κB mediates the activation of GADD45g by oncogene inhibitors JQ1 and AC220. qRT-PCR analysis of GADD45g expression in AML cell lines (A) or in BMMNCs from patients with AML (B) (n = 6) treated with 5 nM romidepsin (Romi) for 48 hours. Molm-13 cells (C) and THP-1 cells (D) were treated with or without 3 nM Romi for 48 hours. The cells were subjected to ChIP analysis by using antibodies against acetyl-histone H3 (Lys9) and anti–acetyl-histone H4 (Lys8). The enriched DNA that associated with the promoter region of GADD45g was quantified by using qPCR. (E) qRT-PCR and western blot analysis of Gadd45g expression in 32D cells transduced with FLT3-ITD, MLL-AF9, or control constructs. qRT-PCR analysis of GADD45g expression in Molm-13 cells (F), THP-1 cells (G), and MV4-11 cells (H) treated with Romi (3 nM), AC220 (10 nM), JQ1 (100 nM), or combinations as indicated for 48 hours. (I) qRT-PCR analysis of GADD45g expression in AML cell lines treated with NF-κB inhibitors PDTC or BAY 11-70825 for 24 hours at the doses indicated. (J) The AML cell lines were treated with dimethyl sulfoxide (DMSO), JQ1 (100 nM), or AC220 (10 nM) for 24 hours and then subjected to western blot to detect the indicated proteins. (K) The AML cell lines were treated with DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. Representative immunofluorescence (left) showing the localization of NF-κB p65 from 3 independent experiments, and quantification (right) of the mean fluorescence intensity in nuclear. Scale bars, 5 μm. (L) Relative activity of NF-κB–dependent luciferase reporter in the indicated AML lines treated with either DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. (M) The AML cell lines were treated with DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. The cells were subjected to ChIP analysis using antibodies against NF-κB p65. The enriched DNA that associated with the promoter region of GADD45g was quantified by using qPCR. Data are presented as the mean ± SD of ≥3 independent experiments, and comparisons were evaluated by using the 2-tailed Student t test. Multiple groups were analyzed with the 1-way analysis of variance. *P < .05, **P < .01, ***P < .001, ****P < .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-IKK, phosphorylated IKK; NS, not significant; shCtrl, scramble small hairpin RNA; shGG, GADD45g small hairpin RNA.

Epigenetic regulation and relevant leukemic oncogenes repress GADD45g expression in AML, and NF-κB mediates the activation of GADD45g by oncogene inhibitors JQ1 and AC220. qRT-PCR analysis of GADD45g expression in AML cell lines (A) or in BMMNCs from patients with AML (B) (n = 6) treated with 5 nM romidepsin (Romi) for 48 hours. Molm-13 cells (C) and THP-1 cells (D) were treated with or without 3 nM Romi for 48 hours. The cells were subjected to ChIP analysis by using antibodies against acetyl-histone H3 (Lys9) and anti–acetyl-histone H4 (Lys8). The enriched DNA that associated with the promoter region of GADD45g was quantified by using qPCR. (E) qRT-PCR and western blot analysis of Gadd45g expression in 32D cells transduced with FLT3-ITD, MLL-AF9, or control constructs. qRT-PCR analysis of GADD45g expression in Molm-13 cells (F), THP-1 cells (G), and MV4-11 cells (H) treated with Romi (3 nM), AC220 (10 nM), JQ1 (100 nM), or combinations as indicated for 48 hours. (I) qRT-PCR analysis of GADD45g expression in AML cell lines treated with NF-κB inhibitors PDTC or BAY 11-70825 for 24 hours at the doses indicated. (J) The AML cell lines were treated with dimethyl sulfoxide (DMSO), JQ1 (100 nM), or AC220 (10 nM) for 24 hours and then subjected to western blot to detect the indicated proteins. (K) The AML cell lines were treated with DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. Representative immunofluorescence (left) showing the localization of NF-κB p65 from 3 independent experiments, and quantification (right) of the mean fluorescence intensity in nuclear. Scale bars, 5 μm. (L) Relative activity of NF-κB–dependent luciferase reporter in the indicated AML lines treated with either DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. (M) The AML cell lines were treated with DMSO, JQ1 (100 nM), or AC220 (10 nM) for 24 hours. The cells were subjected to ChIP analysis using antibodies against NF-κB p65. The enriched DNA that associated with the promoter region of GADD45g was quantified by using qPCR. Data are presented as the mean ± SD of ≥3 independent experiments, and comparisons were evaluated by using the 2-tailed Student t test. Multiple groups were analyzed with the 1-way analysis of variance. *P < .05, **P < .01, ***P < .001, ****P < .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-IKK, phosphorylated IKK; NS, not significant; shCtrl, scramble small hairpin RNA; shGG, GADD45g small hairpin RNA.

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