Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 1. Synergistic antineoplastic effects of azacytidine and erlotinib against SKM1 cells. SKM1 cells were cultured in control conditions (DMSO) or in the presence of the indicated concentrations of azacytidine (AZA) and erlotinib (ERLO), alone or in combination, for (a) 24 h or (b) 48 h, and then assessed for viability by a colorimetric MTS conversion assay. The reduction in MTS conversion is depicted on a 0 (control conditions) to 1 (no conversion) scale. Results are means of duplicate assessments from one out of three independent experiments. (c) In addition, the dose–response curve of each drug was determined and combination index (CI) values for varying AZA/ERLO concentration ratios (4:1, 1:1 and 1:4) were calculated according to the Chou–Talalay’s method at the 24 h time point, with the biological response being expressed as the fraction of affected cells. Dotted lines represent 95% confidence intervals of each condition. The fraction of affected cells for which the combination of AZA and ERLO at the indicated ratio begins to exert synergistic effects is indicated.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Cell Culture, Control, Concentration Assay

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 2. Synergistic killing of leukemic cells by azacytidine plus erlotinib. The indicated cell lines (all originally established from MDS or AML patients) were maintained in control conditions (DMSO) or treated for 72 h with 1 mM azacytidine (AZA, A), 1 mM decitabine (DEC, D), 10 mM erlotinib (ERLO, E), 10 mM gefitinib (GEFI), 0.5 mM sunitinib (SUNI), 0.5 mM dasatinib (DASA), 1 mM sorafenib (SORA), 5 mM imatinib (IMA) and 5 mM lapatinib (LAPA), alone or combined as indicated, followed by cytofluorometry for the assessment of apoptosis-associated variables upon co-staining with the Dcm-sensitive fluorochrome DiOC6(3) and the exclusion dye PI. (a) Representative dot plots of SKM1 cells (numbers indicate the percentage of cells found in each quadrant). (b, c) Black and white columns illustrate the percentage of dead (PI þ) and dying (PI DiOC6(3)low) cells, respectively (means±s.e.m., n ¼ 3). *Po0.05, **Po0.01, ***Po0.001 (ANOVA plus Bonferroni’s post-hoc test) compared with DMSO-treated cells; #Po0.05, ##Po0.01, ###Po0.001 (ANOVA plus Bonferroni’s post-hoc test) compared with AZA-treated cells; wwPo0.01, wwwPo0.001 (ANOVA plus Bonferroni’s post-hoc test) compared with (b) ERLO-treated or (c) tyrosine kinase inhibitor-treated cells.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Control, Staining

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 3. Biochemical and morphological hallmarks of apoptosis in SKM1 cells succumbing to azacytidine plus erlotinib. SKM1 cells were maintained in control conditions (DMSO) for 24 h or exposed, for the same time, to 1mM azacytidine (AZA, A), 10mM erlotinib (ERLO, E), 50mM Z-VAD-fmk (Z-VAD), 5mM HA14-1, 0.5mM ABT-263 and 0.1mM obatoclax (Oba), alone or in the indicated combinations. Thereafter, cells were either processed for the immunoblotting-assisted detection of (a) caspase-3 (CASP-3) maturation, (b) PARP cleavage, and (h) the abundance of multiple Bcl-2 family members, either subjected to immunofluorescence microcopy for the visualization of (c, d) cytochrome c (Cyt c) subcellular localization and (c, e) nuclei or (f, g, i) analyzed by cytofluorometry upon co-staining with the Dcm-sensitive fluorochrome DiOC6(3) and the exclusion dye PI. Representative immunoblots are shown in a, b and h. b actin levels were monitored to ensure equal loading of lanes. Quantitative data for relevant proteins are reported in Supplementary Figure S1. In c, representative immunofluorescence microphotographs are shown (bars¼ 2.5 or 10 mm, as indicated), while quantitative data (means±s.e.m., n ¼ 3) on nuclear karyorrhexis and Cyt c release are illustrated in d and e, respectively. Panel f depicts representative dot plots (numbers indicate the percentage of cells found in each quadrant). In g and i, black and white columns illustrate the percentage of dead (PIþ) and dying (PIDiOC6(3)low) and cells, respectively (means±s.e.m., n ¼ 3). ***Po0.001 (ANOVA plus Student’s t-test) compared with A þ E-treated cells in the absence of Z-VAD (d, e and g); ##Po0.01, ###Po0.001 (ANOVA plus Student’s t-test) compared with AZA-treated cells (i); wwPo0.01, wwwPo0.001 (ANOVA plus Student’s t-test) compared with ERLO-treated cells (i).

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Control, Western Blot, Staining

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 4. DNA damage foci induced by the combination of azacytidine and erlotinib. SKM1 cells were cultured for 24 h in control conditions (DMSO) or in the presence of 1 mM azacytidine (AZA, A), 10 mM erlotinib (ERLO, E), 50 mM Z-VAD-fmk (Z-VAD), alone or combined. Thereafter, cells were processed for the immunofluorescence microscopy-assisted quantification of the levels (none, low, intermediate or high) of nuclear gH2AX þ and 53BP1 þ foci. (a) Representative microphotographs are shown (bar ¼ 5 mm); (b) quantitative data are reported (means±s.e.m., n ¼ 3). Alternatively, the intensity of H2AX phosphorylation was determined (d, e) by cytofluorometry upon staining with a gH2AX-specific antibody or (f) by immunoblotting. Results from one representative experiments out of three are reported in d and f (b actin levels were monitored to ensure equal loading of lanes), while quantitative cytofluorometric data, upon normalization to control conditions, are illustrated in e (means±s.e.m., n ¼ 3). *Po0.05, **Po0.01 (ANOVA plus Bonferroni’s post-hoc) compared with DMSO-treated cells; #Po0.05 (ANOVA plus Bonferroni’s post-hoc) compared with AZA-treated cells; ns ¼ non-significant (ANOVA plus Bonferroni’s post-hoc) compared with cells treated with the same chemicals in the absence of Z-VAD. MFI, mean fluorescence intensity.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Cell Culture, Control, Microscopy, Phospho-proteomics, Staining, Western Blot

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 5. Effects of azacytidine and erlotinib on differentiation and cell-cycle progression. (a) SKM1 cells were maintained for 72 h in control conditions (DMSO) or exposed, for the same time, to 0.5 mM azacytidine (AZA, A) and 5 mM erlotinib (ERLO, E), alone or in combination, and then processed for May-Gru¨nwald–Giemsa staining. Representative cytological images are illustrated (bar ¼ 5 mm). Alternatively, SKM1 cells were kept in control conditions (DMSO) or treated with 1 mM AZA, 10 mM ERLO and 50 mM Z-VAD-fmk (Z-VAD), alone or in combination, for 24 h. Thereafter, cells were either (b) processed for the immunoblotting-assisted detection of p21WAF1 and p27KIP1 levels, or subjected to cytofluorometric determinations of (c, d) the cell-cycle distribution and (e, f) of the percentage of cells exhibiting active DNA synthesis. Results from one representative experiment out of three are reported in b (b actin levels were monitored to ensure equal loading of lanes), c (numbers indicate the percentage of cells with a hypodiploid DNA content) and e (numbers indicate the percentage of cells incorporating 5-ethynyl-20-deoxyuridine (EdU)). Quantitative data (means±s.e.m., n ¼ 3) are reported in d and f. ##Po0.01, ###Po0.001 (ANOVA plus Bonferroni’s post-hoc) compared with AZA-treated cells; wwwPo0.001, ns ¼ non-significant (ANOVA plus Bonferroni’s post-hoc) compared with ERLO-treated cells.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Control, Staining, Western Blot, DNA Synthesis

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 6. Impact of azacytidine and erlotinib on the number and size of colonies formed by SKM1 cells. SKM1 cells were seeded in a methylcellulose-based medium containing DMSO (control condition), 0.15 mM azacytidine (AZA, A) or 2.5 mM erlotinib (ERLO, E), alone or in combination, and allowed to generate colonies for 14 days. At the end of the assay, the number and volume of colonies were quantified. (a) Representative plates and colonies (phase contrast microscopy) are shown (bars ¼ 200 mm or 1 cm, as indicated). (b, c) Quantitative data upon normalization to control conditions (means±s.e.m., n ¼ 3) on colony number and volume are reported. ###Po0.001 (ANOVA plus Bonferroni’s post-hoc) compared with AZA-treated cells; wPo0.05, wwPo0.01 (ANOVA plus Bonferroni’s post-hoc) compared with ERLO-treated cells.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Control, Microscopy

Journal: Oncogene

Article Title: Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia.

doi: 10.1038/onc.2012.469

Figure Lengend Snippet: Figure 7. Erlotinib potentiates the cytotoxicity of azacytidine by aggravating its intracellular accumulation. (a) SKM1 cells were incubated for 1 h with 10 nM [3H]-azacytidine ([3H]-AZA) alone (CTRL) or combined with 10 mM erlotinib (ERLO), 10 mM gefitinib (GEFI), 0.5 mM dasatinib (DASA), 50 mM verapamil (VERA, V), 1 mM cyclospor- ine A (CSA, C), 10 mM MK-571 (M) or 50 mM VERA þ 1 mM CSA þ 10 mM MK-571. Thereafter, cells were lysed and radioactivity measured in a liquid scintillation counter. Columns report intracellular [3H]- azacytidine levels (means±s.e.m., n ¼ 3) upon normalization to control conditions. #Po0.05, ###Po0.001 (ANOVA plus Dunnett’s test) compared with [3H]-AZA-treated cells. (b) SKM1 cells were treated with 10 mM ERLO alone or combined with 1 mM azacytidine (AZA) for 1 h, then processed for the quantification of intracellular ERLO by mass spectrometry. Columns report intracellular ERLO levels (means±s.e.m., n ¼ 3). ns ¼ non significant (ANOVA plus Student’s t-test) compared with ERLO-treated cells. (c) SKM1 cells were left untreated or treated with 1 mM AZA, 10 mM ERLO or 1 mM AZA þ 10 mM ERLO, alone or combined with 50 mM VERA þ 1 mM CSA þ 10 mM MK-571 for 48 h, followed by cytofluorometry for the assessment of apoptosis-associated variables upon co-staining with the Dcm-sensitive fluorochrome DiOC6(3) and the exclusion dye PI. Black and white columns illustrate the percentage of dead (PI þ) and dying (PI DiOC6(3)low) cells, respectively (means±s.e.m., n ¼ 3). ###Po0.001 (ANOVA plus Student’s t-test) compared with AZA- treated cells.

Article Snippet: The SKM1 cell line was a kind gift of Patrick Auberger (INSERM U895, Nice, France), while MOLM-13, KG-1, Kasumi-1, HL-60 and MV411 cells were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Incubation, Radioactivity, Control, Mass Spectrometry, Staining