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

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Comparison of gene expression between healthy BM samples (GTEx, n=70) and AML samples (TCGA, n=173) using Gepia . ITGA6 and ITGA7 are overexpressed in AML. (B) Flow cytometry analysis of laminin receptor expression on healthy mobilized CD34+ CD38-HSPC (n=5), primary CD33+ AML cells (n=60) and AML cell lines (n=7) shows an overexpression of integrin α6 and α7 in AML, depicted as mean +/-SD. (C) Exemplary flow cytometry staining of two selected patients showing laminin receptor surface expression on primary AML cells.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Comparison, Gene Expression, Flow Cytometry, Expressing, Over Expression, Staining

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Flow cytometric analysis shows heterogeneous laminin receptor surface expression between AML cell lines. HNT-34, SKM-1 and THP-1 display the expression of the most laminin receptors as opposed to MOLM-13, which is negative for all laminin receptors analyzed. (B) Western blotting of laminin receptors confirms the expression patterns of integrin α3, α6 and BCAM as seen by flow cytometry. Expression of integrin α7 is very pronounced for Kasumi-1 in Western blotting, but not measurable via flow cytometry. (C) ImageStream analysis of intracellular and extracellular integrin α7 shows expression on the cell surface in SKM-1, but only intracellular expression in Kasumi-1. (D) Comparison between laminin receptor expression on BM and PB primary AML cells reveals higher integrin α7 expression on PB cells. (E) Comparison between laminin receptor expression on AML samples positive for CD34 (> 5 % CD34+ cells) and negative for CD34 (< 1 % CD34+ cells) shows higher surface expression of integrin α7 on CD34-AML samples, whereas integrin α6 and BCAM are higher expressed in CD34+ AML.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Expressing, Western Blot, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: (A) Representative images showing adhesion assays with primary AML cells and the AML cell line THP-1 on different laminin isoform coatings. Plastic dishes were coated with single laminin isoform droplets and unspecific cell adhesion to plastic was blocked with BSA. Laminin specific adhesion is seen in the circular droplet region. Brightfield images were acquired with a microscope (Zeiss, Primovert). (B) Schematic drawing of laminin-511 and its binding specificities. All four laminin receptors have been described to bind to the C-terminal end of laminin-511 ( , ) (C) Phenotyping of adherent and non-adherent cell fractions, marker expression on adherent cells was normalized to the expression on non-adherent cells within each sample. Adherent cells show a higher expression of integrin α3 and α6 and a reduced expression of NKG2DL. (D) Primary AML cells adhere to laminin-511 but not to laminin-211 or laminin-111. (E) Proliferation assays with the SKM-1 AML cell line on different laminin coatings or without coating (control). Laminin-211 coating slightly increases cell proliferation (n=3). (F) Blocking antibodies against integrin α6 and integrin β1 reduce adhesion of primary AML cells to laminin-511 whereas blocking antibodies against integrin α7 and BCAM have no effect on cell adhesion.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Microscopy, Binding Assay, Marker, Expressing, Control, Blocking Assay

Journal: bioRxiv

Article Title: Laminin Receptor Characterization in Acute Myeloid Leukemia: Integrin α7β1 Defines non-Leukemic Stem Cells with Migratory Potential

doi: 10.1101/2024.03.29.587290

Figure Lengend Snippet: Representative images of adhesion assays to different laminin isoform coatings. (A) Adhesion assays of AML cell lines show strong adhesion to laminin-511 and weak adhesion to laminin-332. All tested cell lines but MOLM-13 adhere to laminin-511. (B) Adhesion assays of primary AML cells show adhesion to laminin-511 in some samples, while other patient samples do not adhere. (C) Gene knockouts of integrin α6 and BCAM were generated in the AML cell lines Kasumi-1 and SKM-1 and the absence of surface expression of the respective marker was validated using flow cytometry. Deletion of integrin α6 reduces adhesion to laminin-511, but deletion of BCAM does not affect adhesion. (D) Flow cytometry data of primary AML samples analyzed for integrin α6 and α7 co-expression in non-LSC (NKG2DL+) and LSC populations (NKG2DL-). (E) QRT-PCR analysis of ITGA7 isoforms in primary AML cells (n=11) and AML cell lines (n=7). The isoform α7X2 is higher expressed than α7X1.

Article Snippet: AML cell lines (Kasumi-1, MOLM-13, NOMO-1, SKM-1, THP-1) were obtained from DSMZ and human umbilical cord endothelial cells (HUVEC) from ATCC.

Techniques: Generated, Expressing, Marker, Flow Cytometry, Quantitative RT-PCR