Article Figures & Data
Figures
- Figure 1.
Action of LBH589 on acetylation of histone H4 in myeloma cells and on the proliferation of multiple myeloma cells. A, dose and time effect of LBH589 on histone H4 acetylation in myeloma cells. MM1S cells were treated for the indicated times with LBH589 (100 nmol/L; left) and doses for 24 hours with LBH589 (right), and acetylation of histone H4 was detected by Western blotting with anti-acetyl-histone H4 antibody. As a loading control, an anti-actin Western blot was done. B, action of different antimyeloma agents on histone H4 acetylation. Cells were treated with doxorubicin (1 μmol/L), melphalan (50 μmol/L), bortezomib (10 nmol/L), or LBH589 (100 nmol/L) for the indicated times, and acetylated histone H4 was analyzed by Western blotting. C, MTT uptakes of myeloma cell lines incubated with different doses of LBH589. Cells were plated at identical densities in 48-well dishes, and LBH589 was added at the indicated concentrations. MTT uptake assays were done 48 hours later as described in Materials and Methods. The average proliferation values of control untreated samples were taken as 100%. Points, mean of quadruplicates of an experiment that was repeated at least twice; bars, SD. D, time course and dose effect of LBH589 on MM1S cells. Cells were plated at identical densities in 48-well dishes, and LBH589 was added at the indicated concentrations. MTT uptake assays were done 24, 48, 72, and 96 hours later as described in Materials and Methods as described above.
- Figure 2.
LBH589 causes death of patient cells with multiple myeloma and potentiates the antimyeloma action of bortezomib, dexamethasone, and melphalan. A, action of LBH589 in cells from patients with multiple myeloma. Patient cells were plated in six-well plates and treated with LBH589 at 10 and 100 nmol/L. After 18 hours, cells were stained with Annexin V-FITC and four monoclonal antibodies (CD38, CD56, CD28, and CD45), which allows the distinction between myeloma plasma cells and the remaining normal bone marrow cells, normal lymphocytes, and granulocytes. B, LBH589 (3 nmol/L) was combined with bortezomib (2 nmol/L), dexamethasone (1 μmol/L), or melphalan (2.5 μmol/L), and after 48 hours, MTT assays were done on MM1S cells. C, combination of LBH589 with bortezomib enhances cytotoxicity against patient multiple myeloma cells. Patient's multiple myeloma cells were treated with LBH589 (10 nmol/L) and bortezomib (5 nmol/L) for 18 hours, and apoptosis was determined by staining with Annexin V-FITC.
- Figure 3.
A, genes deregulated by LBH589. MM1S cells were treated with LBH589 (100 nmol/L). RNA was isolated, and cDNA was hybridized to oligonucleotide microarrays and incubated as described in Materials and Methods. Genes modified ≥2-fold were grouped according to different functional categories. B, LBH589 causes cell cycle changes in MM1S cells. MM1S cells were incubated with LBH589 (100 nmol/L) for 3, 6, 12, 18, and 24 hours and examined for cell cycle profile using CD38-CD138/propidium iodide double staining. Bottom, percentage of cells in the different cell cycle phases. C, MM1S cell were treated with LBH589 (100 nmol/L) for the indicated times, and the expression of cell cycle–related proteins was analyzed by Western blotting. D, colorgram of cell cycle deregulated genes after 15 hours of LBH589 (100 nmol/L) treatment. Results from two different experiments (Control_1 versus LBH15h_1 and Control_2 versus LBH15h_2). Average fold change for both experiments (right).
- Figure 4.
LBH589 disrupts the mithochondrial outer membrane and causes apoptosis. A, time course experiment with LBH589 showing increasing cell death (Annexin-positive cells). B, LBH589 provokes DNA fragmentation. MM1S cells were treated with LBH589 (100 nmol/L) or bortezomib (10 nmol/L) for the indicated times, and DNA was isolated and analyzed by agarose gel electrophoresis. Position of the Mr markers (right). C, LBH589 induces ΔΨm disruption. MM1S cells were treated with LBH589 (100 nmol/L), and the ΔΨm analyses were done with [DiOC6(3)low] by flow cytometry. D, MM1S were treated for the indicated times with LBH589, and the subcellular distribution of cytochrome c and AIF in mithochondrial and cytosolic fractions was analyzed by Western blotting.
- Figure 5.
Effect of LBH589 on apoptotic pathways. A, MM1S cells were treated with LBH589 (100 nmol/L) for the indicated times, and expression of Apaf-1, caspase-9, caspase-3, and PARP proteins was analyzed by Western blotting of cell extracts using specific antibodies. B, colorgram of genes involved in apoptosis deregulated after 15 hours of LBH589 (100 nmol/L) treatment. C, validation of microarray data by SYBR Green quantitative real-time PCR. Values for each gene were normalized to expression levels of GAPDH. From an experiment representative of at least three independent experiments. D, effect of the pancaspase inhibitor Z-VAD-FMK on LBH589-induced cell death. MM1S cells were plated and pretreated where indicated, with Z-VAD-FMK (50 μmol/L) for 60 minutes. LBH589 (100 nmol/L) or bortezomib (10 nmol/L) were added to the corresponding samples, and the experiment was continued for 24 hours. MTT uptake was carried out as described above. E, gene expression data obtained by quantitative PCR analyses of five genes that participate in the extrinsic apoptotic pathway. Representative of at least three independent experiments. F, activation of caspase-8 and cleavage of Bid induced by LBH589. MM1S cells were treated as in (A), and the cleavage of caspase-8 and Bid was examined by Western blotting.
- Figure 6.
Expression of Bcl-X prevents LBH589-induced cell death. A, action of LBH589 on the levels of Bcl-2 and Bcl-X proteins. MM1S cells were treated with LBH589 (100 nmol/L) for the indicated times, and cell extracts were used for Western blotting with anti-Bcl-2 and anti-Bcl-X antibodies. B, expression of wild-type AU1-Bcl-X in MM1S cells. Cells were infected with retroviruses that included an empty vector, or an AU1-Bcl-X containing vector, and the expression of the exogenous and endogenous Bcl-X protein was analyzed by Western blotting of cell extracts with an anti-Bcl-X antibody. C, action of AU1-Bcl-X expression on LBH589-induced cell death. Control and AU1-Bcl-X-expressing MM1S cells were plated at identical densities, and then LBH589 was added, where indicated. Cell viability was analyzed 24 hours later by MTT assays as described above. Columns, mean of quadruplicates; bars, SD.
Volume 66, Issue 11
