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:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat enters Jurkat cells but not U937 cells or monocytes. Jurkat cells, U937 cells, and monocytes were exposed to HIV Tat (100 ng/ml) for 0 min (lanes 1, 5, and 10), 30 min (lanes 2, 6, and 11), 1 h (lanes 3, 7, and 12), or 3 h (lanes 4, 8, and 13). After the indicated times, cells were collected, cell lysates were separated by SDS-10% PAGE, and cell proteins were transferred to nitrocellulose membranes and immunoblotted with the TR1 murine monoclonal antibody to Tat (57). Lanes 9 and 14 contain recombinant Tat protein.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Recombinant

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat increases TRAIL expression in U937 cells and human monocytes, but Tat toxoid does not. (A) TRAIL expression increases with increasing doses of Tat protein. Monocytes (a) and CD4+ cells (b) were incubated with or without HIV Tat or Tat toxoid (10 to 1,000 ng/ml) at 37°C for 20 h, and 10 μl of cell lysate (6 × 104 cell equivalents) was separated by SDS-10% PAGE. Cell proteins were transferred to nitrocellulose membranes and immunoblotted with polyclonal anti-TRAIL (H-257; 1:1,000). Lanes (from left): 1, no HIV Tat protein; 2 to 5, cell extracts exposed to 10, 50, 100, and 1,000 ng of Tat/ml, respectively. Actin (antibody A-2668; Sigma) was an internal control. Similar results were obtained with fresh human monocytes and with the U937 cell line. TRAIL production in fresh human T cells or in Jurkat cells was not stimulated by exposure to Tat. (B) HIV Tat induces the transcription of TRAIL mRNA in U937 cells and monocytes. Total RNA from U937 cells, monocytes, or CD4+ cells was isolated and subjected to reverse transcription-PCR. β-Actin mRNA was amplified over 25 cycles and TRAIL mRNA was amplified over 35 cycles of PCR. Transcription of control β-actin or TRAIL/APO2L mRNA in U937 cells is shown after 0, 2, 6, 12, or 24 h of incubation in the presence of HIV Tat (100 ng/ml).

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Expressing, Incubation, Isolation, Amplification

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat only slightly increases membrane-associated TRAIL expression in U937 cells and monocytes. U937 cells (A), human monocytes (B), Jurkat cells (C), and primary CD4+ cells (D) were stimulated with HIV Tat (100 ng/ml) for 20 h, stained with PE-conjugated mouse anti-human TRAIL or mouse IgG1 control monoclonal antibody as described in Materials and Methods, and then subjected to flow cytometry. The histograms shown are representative of three independent experiments.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Expressing, Staining, Flow Cytometry

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat increases secretion of soluble TRAIL in media of U937 cells and monocytes. For this experiment, 3 × 106 U937 cells (A), human monocytes (B), and CD4+ cells (C) in 100 μl of medium were incubated with or without HIV Tat (10 to 1,000 ng/ml) at 37°C for 20 h, and then 20 μl of cell medium was separated by SDS-10% PAGE. Proteins were transferred to nitrocellulose membrane and incubated with polyclonal anti-TRAIL (H-257; 1:1,000). Lanes (from left), 1, no HIV Tat protein; 2 to 5, extracellular media exposed to 10 to 1,000 ng of Tat/ml. TRAIL secretion could not be detected from T-lymphocyte cultures after 20 h.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Incubation