Article Figures & Data
Figures
- Fig. 1.
Flavopiridol markedly inhibits activation of NF-κB induced by histone deacetylase inhibitors in human leukemia cells. A, U937 cells were untreated or treated with 1 mM NaB, 100 nM FP, or the combination (FP + NaB) for 24 h, after which nuclear extracts were then prepared and subjected to EMSA as described under Materials and Methods. For C + C′, a 100-fold excess of unlabeled NF-κB oligonucleotides was added to the nuclear extract obtained from untreated cells before the addition of labeled NF-κB oligonucleotides. B, U937 cells were untreated or treated with 1.5 μM SAHA, 100 nM FP, or the combination of FP and SAHA for 24 h. NF-κB DNA binding activity was determined by EMSA as described above. C, for the supershift assay, nuclear extracts were preincubated with specific anti-p50 and anti-p65 antibodies and subjected to EMSA. Results are representative of three separate studies. For all studies, lanes were loaded with 4 μgof protein. NF-κB DNA binding signals were quantified using Scion Image software (Scion Corporation). Bottom, Mean densitometric values from three independent experiments were normalized to controls that were assigned an arbitrary value of 1.0. The data represent the means ± S.D. (n = 3). OD, optical density; NS, nonspecific; C, control.
- Fig. 2.
Inhibition of NF-κB activity in U937 cells transfected with an IκBα super-repressor mutant. U937 cells were stably transfected with an IκBα mutant (Ser32, 36/Ala) or an empty vector [pcDNA (3.1)] as described under Materials and Methods. Two U937 cell clones stably expressing IκBα mutants (designated 2C8 and 2H6) were then isolated and prepared. A, empty vector control cells and the two IκBα mutant transfectants (2C8 and 2H6) were treated with 1 mM NaB, 100 nM FP, or the combination of FP and NaB, after which the nuclear extracts were prepared and subjected to EMSA as described under Materials and Methods. B, empty vector controls and the two IκBα mutant transfectants (2C8 and 2H6) were treated with 1.5 μM SAHA, 100 nM FP, or the combination of FP and SAHA, after which nuclear extracts were prepared and subjected to EMSA analysis as described above. Bottom, densitometric analysis of signals from EMSA analysis shown above. Results represent values compared with vector controls and are representative of three separate studies. NF-κB DNA binding signals were quantified and analyzed as described in Fig. 1. The data are expressed as means ± S.D. (n = 3).
- Fig. 3.
Stable transfection of cells with an IκBα mutant enhances FP/NaB- and FP/SAHA-induced apoptosis in U937 cells. U937 cells were stably transfected with IκBα mutant (Ser32, 36/Ala) or an empty vector (pcDNA3.1) as described under Materials and Methods. U937/IκBα mutant (clones 2C8 and 2H6) and /pcDNA3.1 cells were treated with 100 nM FP, the histone deacetylase inhibitors NaB (1 mM) or SAHA (1.5 μM), or the combination of FP/NaB (A) and FP/SAHA (B) for 24 h. Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry as described under Materials and Methods. The values obtained from the Annexin V assay represent the means ± S.D. for three separate experiments. ⋆⋆, values for U937/IκBα mutant cells (clones 2C8 and 2H6) significantly greater than those for empty vector controls (pcDNA3.1 cells) by the Student's t test; p < 0.01. ⋆⋆⋆, p < 0.001.
- Fig. 4.
Stable transfection of cells with an IκBα enhances FP/NaB- and FP/SAHA-induced mitochondrial injury in U937 cells. U937/IκBα mutant (clones 2C8 and 2H6) and /pcDNA3.1 cells were treated with 100 nM FP, NaB (1 mM) or SAHA (1.5 μM), or the combination of FP/NaB (A) and FP/SAHA (B) for 24 h. The cytosolic S-100 fractions were prepared and subjected to Western blot assay using antibodies against cytochrome c (cyto c) and AIF. Each lane was loaded with 20 μg of protein; blots were subsequently stripped and reprobed with antibodies directed against β-actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results.
- Fig. 5.
Stable transfection of cells with an IκBα enhances FP/NaB- and FP/SAHA-induced caspase activation in U937 cells. U937/IκBα mutant (clones 2C8 and 2H6) and /pcDNA3.1 cells were treated with 100 nM FP, NaB (1 mM) or SAHA (1.5 μM), or the combination of FP/NaB (A) and FP/SAHA (B) for 24 h. Total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP, C-caspase-3, caspase-8, caspase-9, and β-actin as indicated. Each lane was loaded with 20 μg of protein; blots were subsequently stripped and reprobed with antibodies directed against β-actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results. C-caspase-3, cleaved caspase 3; CF, cleavage fragment.
- Fig. 6.
Stable transfection of RelA/p65 siRNA enhances FP/NaB- and FP/SAHA-induced apoptosis in U937 cells. U937 cells were stably transfected with RelA/p65 siRNA or an empty vector (3.1-H1 hygro) as described under Materials and Methods. A, comparison of the expression of RelA/p65 in U937/p65 siRNA and U937/3.1-H1 hygro cells is shown by Western blot. U937/p65 siRNA (p65) and /3.1-H1 hygro (3.1) cells were treated with FP (100 nM), NaB (1 mM), or SAHA (1.5 μM), or the combination of FP/NaB (B) and FP/SAHA (C) for 24 h. Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. The values obtained from Annexin V assays represent the means ± S.D. for three separate experiments. ⋆⋆, values for RelA/p65 siRNA cells significantly greater than those for empty vector controls (3.1) by the Student's t test, p < 0.01.
- Fig. 7.
Stable transfection with an IκBα mutant enhances FP/NaB and FP/SAHA-induced activation of phosphor-JNK (P-JNK) and down-regulation of XIAP, p21CIP1/WAF1, and Mcl-1. A, U937/IκBα mutant (2C8 and 2H6) and /pcDNA3.1 cells were treated with 1 mM NaB, 100 nM FP, or the combination of FP/NaB. B, transfected cells were also treated with 1.5 μM SAHA, 100 nM FP, or the combination of FP/SAHA. Total cellular extracts were prepared and subjected to Western blot assay using antibodies against phosphor-JNK, JNK, XIAP, Mcl-1, p21CIP1/WAF1, p27KIP1, and β-actin. Each lane was loaded with 20 μg of protein; blots were subsequently stripped and reprobed with antibodies to actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results.
In this issue
Contribution of Disruption of the Nuclear Factor-κB Pathway to Induction of Apoptosis in Human Leukemia Cells by Histone Deacetylase Inhibitors and Flavopiridol
