Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Cytarabine dose-dependently decreases cell number and induces differentiation. ( A ) DNA damage pathway activation by pyrimidine synthesis inhibitors and cytarabine. ( B ) U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM). The number of viable cells and the expression of differentiation markers were determined after 72 h. Mean fluorescence intensity (MFI) of CD11b and CD64 was calculated as described under “ ” section. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t -test) compared with control (ctrl). ( C ) Representative histograms (out of three independent flow cytometric analyses shown in B) with black line representing isotypic control and red line representing the expression of CD11b. ( D ) Morphological analysis of U937 cells treated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10 and 100 nM). May-Grünwald-Giemsa stained cytospin preparations (100 × magnification). (E) Respiratory burst in U937 cells treated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM) for 72 h. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Activation Assay, Incubation, Expressing, Fluorescence, Control, Staining

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Cytarabine induces cell cycle arrest and activates Chk1. U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM). ( A ) Representative histograms of propidium-labelled cells from three independent experiments analyzed by flow cytometry. ( B, C ) Pan-caspase inhibitor Z-VAD-FMK (10 μM) was added 30 min before the addition of agents. ( B ) The representative dot plots of cells stained with annexin V-FITC/PI and analyzed by flow cytometry. ( C ) The percentage of annexin V-FITC-positive cells, the number of viable cells and the expression of differentiation markers were determined after 72 h. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl). ( D ) U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM) for 48 h (upper panels) or 72 h (lower panels). Total cell lysates were isolated after 48 and 72 h and analyzed by Western blotting for the level of Ser-345-phosphorylated Chk1, total Chk1, Tyr-15-phosphorylated CDC2 and total CDC2. Representative immunoblots from three independent experiments are shown.

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Incubation, Flow Cytometry, Staining, Expressing, Control, Isolation, Western Blot

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Pharmacological inhibition of ATR/Chk1 pathway prevents differentiation and cell cycle arrest. U937 cells were grown in the presence of increasing concentrations of AraC (10, 100 nM). Torin2 (100 nM), VE-821 (10 µM) or vehicle (DMSO) were added 30 min before the addition of AraC. (A) Total cell lysates were isolated after 48 h and analyzed by Western blotting for the level of Ser-345-phosphorylated Chk1, total Chk1, Tyr-15-phosphorylated CDC2 and total CDC2. Representative immunoblots from three independent experiments are shown. ( B – C ) The number of viable cells, the expression of differentiation markers and the cell cycle progression were determined for Torin2 (B) and VE-821 (C) pre-treated cells as described under “ ” section. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t -test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Inhibition, Isolation, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Down-regulation of Chk1 reduces the effects of cytarabine on the expression of differentiation markers and S-phase arrest. U937 cells were transfected with siRNA against CHK1, and respective nontargeting siRNA was used as a negative control. AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (100 nM) were added 24 h after transfection. ( A ) Total cell lysates were isolated 3 or 48 h after the addition of agents and analyzed by Western blotting for the level of Chk1. Western blot analyses are shown for each of the three independent experiments. ( B ) The number of viable cells, the expression of differentiation markers and the cell cycle progression were analyzed by flow cytometry 72 h after addition of agents. ( C ) Representative histograms of propidium-labeled cells analyzed by flow cytometry. ( D ) Percentage of cells in G 0 /G 1 , S, and G 2 /M-phases of the cell cycle. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Expressing, Transfection, Negative Control, Isolation, Western Blot, Flow Cytometry, Labeling, Control

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

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 expression is remarkably up-regulated in AML. A. Heat map showed the differentially expressed circRNAs (bone marrow mononuclear cells of AML patients v.s. bone marrow mononuclear cells of healthy controls, GSE94591), and the filter criteria was P < 0.05,Log2(Change fold)> 2.5. B. The expression of circ_0004277 in bone marrow mononuclear cells of AML patients was significantly down-regulated compared with that of healthy controls (GSE94591). C. qRT-PCR showed that circ_0004277 expression was lower in the bone marrow samples of 37 AML patients than that in 37 healthy controls. D. qRT-PCR revealed that circ_0004277 expression in AML cells (KG-1a, THP-1, K562 and U937) was down-regulated compared with the human normal stromal cells (HS-5). E. qRT-PCR showed that circ_0004277 was resistant to RNase R. ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 directly targets miR-134-5p. A. Bioinformatics was employed to predict the binding site between circ_0004277 and miR-134-5p. B. Dual-luciferase reporter gene assay assays demonstrated that miR-134-5p could negatively regulate the luciferase activity of circ_0004277-WT, rather than circ_0004277-MUT in U937 and KG-1a cells. C. RIP assay indicated that circ_0004277 and miR-134-5p were enriched in anti-Ago2 group, suggesting circ_0004277 and miR-134-5p were directly interacted. D. RNA pull-down assay showed that circ_0004277 and miR-134-5p could combine with each other. E. qRT-PCR indicated that miR-134-5p expression was decreased in the circ_0004277 overexpression plasmid group compared with the empty plasmid group. F. qRT-PCR indicated that miR-134-5p expression was increased in the bone marrow samples of 37 AML patients compared with that of 37 healthy donors. G. circ_0004277 expression in AML tissues was negatively correlated with miR-134-5p expression in bone marrow samples of the patients with AML. ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Activity Assay, Pull Down Assay, Quantitative RT-PCR, Expressing, Over Expression, Plasmid Preparation

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: MiR-134-5p inhibits the proliferation and aggressiveness of KG-1a and U937 cells. A. qRT-PCR showed that miR-134-5p expression was up-regulated in AML cell lines (KG-1a, THP-1, K562 and U937) compared with the human normal stromal cells (HS-5). B. qRT-PCR revealed that miR-134-5p expression was down-regulated in U937 and KG-1a cells transfected with miR-134-5p inhibitor. C. CCK-8 assay showed the viability of KG-1a and U937 cells was significantly decreased after the transfection of anti-miR-134-5p. D. Transwell assay indicated that the migration and invasion of KG-1a and U937 cells were decreased after the transfection of anti-miR-134-5p. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Transwell Assay, Migration

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 modulates the biological behaviors of AML cells by sponging miR-134-5p. A. qRT-PCR showed that the miR-134-5p mimics have no effect on the expression of circ_0004277. B. qRT-PCR indicated that miR-134-5p mimics could up-regulate the expression of miR-134-5p. C. CCK-8 assay showed that miR-134-5p mimics could reverse the effect of circ_0004277 overexpression plasmid on the viability of U937 and KG-1a cells. D. Transwell assay showed that the migration and invasion of U937 and KG-1a cells transfected with circ_0004277 overexpression plasmid was reversed by miR-134-5p mimics. ** P < 0.01, *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Over Expression, Plasmid Preparation, Transwell Assay, Migration, Transfection

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 2. Clonal DOT-cell reactivity against AML cells. A and B, In vitro killing of AML KG-1 cells by DOT-cell clones generated from single Vd1þ T cells sorted from healthy donors. Cells were coincubated for 3 hours at 10:1 (E:T) ratio and then analyzed by Annexin V staining (percentage of positive events among prelabeled KG-1 cells). Each bar represents killing of KG-1 cells upon coincubation with individual clones. Dashed red line represents the mean basal tumor cell death (without DOT cells). In B, either anti-Vd1þ TCR-specific mAb or isotype control was added to the cultures. Shown are the clones where the blockade led to clearer reduction in KG-1 targeting. Data represent the average of two technical replicates and are derived from 4 independent healthy donors (HD). C, Real-time PCR assessing B7-H6 mRNA in parental (B7-H6þ/þ) and CRISPR/Cas9-manipulated (B7-H6/) AML HEL cell lines. D, In vitro killing of B7-H6þ/þ or B7-H6/ AML HEL cells by bulk DOT cells produced from 3 healthy donors (tested in technical duplicates). Cells were coincubated for 3 hours at 10:1 (E:T) ratio and then analyzed by Annexin V staining (shown are percentages of positive events among prelabeled HEL cells). Indicated are mean þ SEM (, P < 0.001).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: In Vitro, Clone Assay, Generated, Staining, Control, Derivative Assay, Real-time Polymerase Chain Reaction, CRISPR, Produced

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 3. DOT cells target multiple AML cell types but not healthy leukocytes. In vitro killing assays with DOT cells produced from 3–4 healthy donors, coincubated for 3 hours at 10:1 (E:T) ratio with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from the peripheral blood (C). In A, the dashed red line represents the mean basal tumor cell death; and in B, CTR refers also to tumor cells alone (without DOT cells). Experiments were performed with technical triplicates. In vivo AML targeting by DOT cells. DOT cells (3 injections of 2 107 cells, see Supplementary Fig. S5A–S5C) were transferred to NSG mice (n ¼ 6 CTR, 7 DOT-treated mice) preinjected with KG-1 AML cells (D–E); or NSGS mice (n ¼ 5 CTR, 5 DOT-treated mice) bearing primary AML cells (F–G; patient-derived xenograft, PDX). Tumor burden was assessed in the blood and liver one week after the last DOT-cell transfer (D); or through weekly bleedings (F). Indicated are mean þ SEM, , P < 0.05; , P < 0.001; , P < 0.0001. Animals were sacrificed when advanced disease symptoms (such as back leg paralysis) were observed. Survival curves are presented in panels E (P < 0.05) and G (P < 0.01).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: In Vitro, Produced, In Vivo, Derivative Assay

Journal: Cancer Immunology Research

Article Title: Broad Cytotoxic Targeting of Acute Myeloid Leukemia by Polyclonal Delta One T Cells

doi: 10.1158/2326-6066.cir-18-0647

Figure Lengend Snippet: Figure 4. DOT cells (re-)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. A, DOT cells and standard AML chemotherapy (doxorubicin plus cytarabine) protocols were tested against chemotherapy-na€ve (wild type, wt) or chemo-relapsed (CR, regrown after >99% HEL cell elimination) AML cells. Shown are the percentages of Annexin Vþ HEL cells after 3 hours of treatment. B, Number of AML HEL cells before and after 72 hours of treatment with DOT cells (at 5:1 E:T ratio). Surviving cells (<1%) were resorted and allowed to regrow, thus generating the DOT-treated (DT) samples of (C–E). C, DOT cells were coincubated for 3 hours with nontreated (NT) or previously DOT-treated (DT) AML HEL cells at 5:1 or 10:1 (E:T) ratios. Shown are the percentages of Annexin Vþ HEL cells. D, Number of barcoded AML single-cell lineages in non-treated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. E, Pearson correlation for distribution of barcoded AML single-cell lineages between different treatments. Red, yellow, and green dashed lines represent low, medium, and high correlations, respectively. Indicated are mean þ SEM (, P < 0.01; , P < 0.001; , P < 0.0001).

Article Snippet: AML cell targeting in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) were obtained from and authenticated by the German Resource Center for Biologic Mate- rial (DSMZ); and used at passages p3–p8.

Techniques: Comparison, In Vitro, Activity Assay