Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 1. UNBS1450 induces apoptotic cell death in U937 cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantification (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Staining, Cell Cycle Assay, Incubation, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 2. Na+/K+-ATPase subunit a1 mRNA quantification. Na+/K+-ATPase subunit a1 mRNA content of untreated PBMCs and a wide panel of hematological cancer cell lines including K562, Jurkat and U937 cells was transcribed and then quantified by RT-PCR. The quantification of three independent experiments is expressed in brute 2^DCt values S.D.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 3. (A) Caspase activation. U937 cells were incubated in RPMI + 10% FCS UNBS1450 20 nM up to 24 h. Western blot analysis of UNBS1450-induced cleavage of pro-caspases-9, -8, -7 and -3. (B) Analysis of expression levels of anti- apoptotic proteins. UNBS1450-induced expression level alterations of XIAP, Bcl-2 and Mcl-1. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Western Blot, Expressing

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 4. UNBS1450 enables Bak/Bax activation. U937 cells were incubated for 24 h in RPMI + 10% FCS in presence or in absence of UNBS1450. Bak (A.) and Bax (B.) activation status were assessed by using primary antibodies specifically targeting activated forms of Bak (Ab-1; Calbiochem) and Bax (6A7; Santa Cruz). Counterstaining was done by Hoechst staining to assess apoptotic nuclei. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Staining

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 5. Inhibition by UNBS1450 of TNFa-induced NF-kB activation. (A) K562 and (B) Jurkat cells were pretreated with UNBS1450 at various concentrations from 10 to 50 nM and incubated for 2 h, followed by TNFa addition (20 ng/ml) and an additional incubation period of 6 h. Results are represented as the ratio of the measured luminescence of the firefly luciferase vector divided by the measured luminescence of the Renilla plasmid. Untreated cells were used as a negative control, cells treated with TNFa only as a positive control. Results are presented as mean S.D. of 3 individual measurements performed in triplicates. (C) Effect of UNBS1450 on the binding affinity of NF-kB was assessed by an EMSA on the K562 and Jurkat cell lines. The data shown here were representative for three independent experiments with similar results. (D) For supershift/immunodepletion experiments, the nuclear extracts and labelled probes were incubated in the reaction mixture for 30 min on ice prior to a 30 min incubation with 2 mg of anti-p50 or anti-p65 antibodies. SS designates supershifted bands. (E) Jurkat cells were incubated with UNBS1450 (40 nM) for 2 h, followed by a TNFa (20 ng/ml) activation for the indicated time periods. Cytoplasmic and nuclear extracts were prepared, fractionated on a 10% SDS-page gel, transferred to a membrane and then tested for IkBa and p65. Protein loading and purity of nuclear/cytosolic extracts were verified by lamin B and a-tubulin Western blots. Data shown are representative for three independent experiments with similar results. K562 (F), and U937 (G) cells were incubated for 2 h in RPMI + 10% FCS in presence or in absence of various concentrations (10–50 nM) of UNBS1450 before being activated by TNFa during 22 h. After 24 h of incubation IL-8 concentrations in supernatants were measured. Untreated cells served as negative control whereas cells activated by TNFa only were used as a positive control. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Inhibition, Activation Assay, Incubation, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Binding Assay, Immunodepletion, SDS Page, Membrane, Western Blot

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Volcano plot showing sex differences in gene expression in DLBCL. Genes were considered to be differentially expressed when adjusted p -value < 0.05. Upregulated genes are marked in red, downregulated genes are marked in green, and not significant genes are marked in black.

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques: Gene Expression

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Sex differences in gene expression and pathways among molecular subtypes of DLBCL. ( A ), Venn diagram showing the distribution of the differentially expressed genes among the DLBCL subtypes (adj. p -value < 0.05). The table shows the number of samples for each subtype and sex. ( B ), Venn diagram depicting the significant pathways showing a sex difference among the DLBCL subtypes as analyzed by GSEA (adj. p -value < 0.05).

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques: Gene Expression

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Venn diagram showing the number of genes that are differentially expressed in pre- vs. postmenopausal females among the ABC and GCB subtypes and the overlap of differentially expressed genes in pre- (≤52 years of age) vs. post (≥65 years of age)-menopausal females in the ABC and GCB DLBCL subtypes (adj. p -value < 0.05). The table shows the number of DLBCL cases in the ABC and GCB subgroups and in pre- and postmenopausal females, respectively. * This is the explanation (the names) of the five genes that overlap between the GCG and ABC subtypes of female pre- vs. postmenopausal patients.

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques:

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Identification of genes that likely are regulated by estrogens in the ABC and GCB DLBCL subtypes, respectively. ( A ), Venn diagram showing the overlap of differentially expressed genes in the ABC DLBCL subtype. ( B ), Bar graph showing the log2-fold change of genes that are likely regulated by estrogen in the ABC subtype. Upregulated genes are marked in red, and downregulated genes are marked in blue. ( C ), Venn diagram showing the overlap of differentially expressed genes in the GCB DLBCL subtype. ( D ), Bar graph showing the log2-fold change of genes that are likely regulated by estrogens in the GCB subtype. Upregulated genes are marked in red, and downregulated genes are marked in blue.

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques:

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Representative immunohistochemistry staining of DLBCL lymphoma tissue for NR4A2 ( A ) and MUC5B ( B ). Immunohistochemical staining of DLBCL for NR4A2 ( A ) and MUC5B ( B ). Representative staining of samples with low ( left ) and high ( right ) expression, respectively. Black arrows mark examples of expression of NR4A2 and MUC5B in tumor cells, while blue arrows mark expression in non-malignant cells.

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques: Immunohistochemistry, Staining, Immunohistochemical staining, Expressing

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Correlation between NR4A2 expression and overall survival among female ABC DLBCL patients. NR4A2 expression data for each individual female ABC DLBCL and individual survival data were obtained from the EGAD00001003600 data set. Optimal cut point for high vs. low NR4A2 expression was determined using maximally selected rank statistics from the “maxstat” R package. The number of individuals with high and low NR4A2 expression after optimal cut point determination was 55 and 77, respectively. The R packages “survival” and “survminer” were used for the survival analysis. The pink and blue areas show lower 95% to upper 95% CI for low and high NR4A2 expression, respectively. p = 0.008 for the difference in survival between the high and low NR4A2 -expressing individuals.

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques: Expressing

Journal: Cancers

Article Title: Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma—Possible Estrogen Effects

doi: 10.3390/cancers15041298

Figure Lengend Snippet: Inhibition of estrogen synthesis by the aromatase inhibitor letrozole stimulates the growth of U2932 tumors and affects the expression of NR4A and MUC5B . ( A ), Male NSG mice were injected subcutaneously with U2932 cells and treated subcutaneously daily with vehicle or the aromatase inhibitor letrozole (10 μg/mouse). The vehicle group consisted of 7 mice, letrozole group consisted of 8 mice. Tumor size was measured daily. ( B ), RT-qPCR analysis of NR4A1 , NR4A2 , NR4A3 and MUC5B . Results are presented as relative expressions (mean ± SD). An unpaired two-tailed t-test was used for statistical analysis between the two groups (* p < 0.05, ** p < 0.01).

Article Snippet: The human ABC DLBCL cell line U2932 used in this study was obtained from DSMZ (Leibniz Institute, Braunschweig, Germany) and cultured as previously described [ ].

Techniques: Inhibition, Expressing, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Human Molecular Genetics

Article Title: XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation

doi: 10.1093/hmg/ddv052

Figure Lengend Snippet: High levels of XIAP in B-cell lymphoma activate autophagy. ( A ) B cells (WT) and three diffuse large B-cell lymphoma cell lines, SUDHL5, SUDHL8 and SUDHL10 were subjected to immunoblotting with XIAP and actin antibodies. ( B ) The same cell lines were treated with 400 n m bafilomycin A1 and were subsequently subjected to immunoblotting with LC3 and tubulin antibodies. The diffuse large B-cell lymphoma cell lines, SUDHL5 ( C ), SUDHL8 ( D ) and SUDHL10 ( E ), were treated without or with 10 μM embelin (Emb) for 16 h, and treated without or with 400 n m bafilomycin A1 (Baf A1) for the last 4 h of the experiment, and were subsequently subjected to immunoblotting with LC3 and tubulin antibodies. ( F ) Propidium iodide and FITC-conjugated Annexin A5 staining detected by flow cytometry of B cells (WT) SUDHL5, SUDHL8 and SUDHL10 (5, 8 and 10) treated without or with 10 μM embelin (Emb) for 16 h. The percentage of cells positive for both PI and Annexin A5 are shown. Densitometric measurements of LC3-II bands were normalized to the corresponding actin or tubulin bands and are shown in the corresponding histograms. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P -values were determined using one sample t -tests, where controls are set to 100%. See also Supplementary Material, Figure S7 .

Article Snippet: Wild-type B-cells and human diffuse large B-cell lymphoma cell lines (DLBCL) SUDHL5, SUDHL8 and SUDHL10 [obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSMZ)] were cultured at 37°C, 5% CO 2 in 10% FBS, 2 m m l -glutamine and 100 U/ml penicillin/streptomycin supplemented RPMI 1640 (Invitrogen) (see Supplementary Material online ).

Techniques: Western Blot, Staining, Flow Cytometry, Standard Deviation