Journal: Cancer Medicine

Article Title: Induction of autophagy and autophagy‐dependent apoptosis in diffuse large B‐cell lymphoma by a new antimalarial artemisinin derivative, SM 1044

doi: 10.1002/cam4.1276

Figure Lengend Snippet: SM 1044 induces caspase‐dependent apoptosis in DLBCL cell lines. (A) Structural formula of SM 1044. (B) SU ‐ DHL ‐4 cells were treated with the indicated concentrations of SM 1044, DHA , ART , ARS , and ARM for 24 h, respectively. Cell viability was measured by CCK ‐8 and the percent inhibitions were calculated (mean ± SEM, n = 3). (C) SU ‐ DHL ‐4, SU ‐ DHL ‐10, and OCI ‐ LY 3 cells were treated with the indicated concentrations of SM 1044 for 24 h. The cell viability was measured by CCK ‐8 and the percent inhibitions were calculated (mean ± SEM, n = 3). (D) SU ‐ DHL ‐4, SU ‐ DHL ‐10, and OCI ‐ LY 3 cells were treated with the indicated concentrations of SM 1044 for 24 h. Cell apoptosis was detected by flow cytometry using the FITC ‐Annexin V/ PI apoptosis detection kit (mean ± SEM, n = 3). SU ‐ DHL ‐4: 0 μ mol/L versus 0.05 μ mol/L, P = 0.042; 0.1 μ mol/L, P < 0.001; 0.5 μ mol/L, P < 0.001; 1 μ mol/L, P = 0.0061. SU ‐ DHL ‐10: 0 μ mol/L versus 0.1 μ mol/L, P = 0.023; 0.5 μ mol/L, P < 0.001; 1 μ mol/L, P < 0.001; 5 μ mol/L, P < 0.001. OCI ‐ LY 3: 0 μ mol/L versus 0.05 μ mol/L, P = 0.034; 0.1 μ mol/L, P < 0.001; 0.5 μ mol/L, P < 0.001; 1 μ mol/L, P < 0.001. (E) SU ‐ DHL ‐4 cells were treated with SM 1044 for 24 h. Representative electron microscopy photomicrographs are shown. The red arrows point to the apoptosis bodies. (F) The expressions of caspase‐8, ‐9, ‐3, and PARP were detected by western blot in SU ‐ DHL ‐4, SU ‐ DHL ‐10, and OCI ‐ LY 3 cells treated with the indicated concentrations of SM 1044 for the indicated time courses. (G) Apoptosis was measured in SU ‐ DHL ‐4, SU ‐ DHL ‐10, and OCI ‐ LY 3 cells pretreated with caspase inhibitor Z‐ VAD ‐ FMK for 1 h, and then treated with SM 1044 for another 24 h (mean ± SEM, n = 3). SU ‐ DHL ‐4: SM 1044 versus control, P < 0.001; SM 1044 versus Z‐ VAD ‐ FMK plus SM 1044, P < 0.001. SU ‐ DHL ‐10: SM 1044 versus control, P < 0.001; SM 1044 versus Z‐ VAD ‐ FMK plus SM 1044, P < 0.001. OCI ‐ LY 3: SM 1044 versus control, P < 0.001; SM 1044 versus Z‐ VAD ‐ FMK plus SM 1044, P < 0.001. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLBCL cell lines SU‐DHL‐4, SU‐DHL‐10, and OCI‐LY3 were obtained from the French National Institute of Health and Medical Research (INSERM).

Techniques: CCK-8 Assay, Flow Cytometry, Electron Microscopy, Western Blot, Control

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: Expression of CD40 in primary dog B cell lymphomas. (A) A heat map of selected gene expression in dog B-cell and T-cell lymphomas. Expressions of genes, chosen for prototypical expression in B-cells and T-cells, were shown as a heat map where color represents fluorescence intensity of hybridized probes as indicated at the bottom and therefore the amount of gene transcript in any given sample. Generally, Red/Orange represents high expression, Yellow/Green represents moderate expression and Blue/Black represents little or no expression. (B) DLBCL samples were stained for CD22 and CD40 (using CD40L-CD8 fusion protein). A representative result using an isotype control antibody and streptavidin (SA control) is shown as a negative control (left panel). One representative sample of 3 different primary dog B-cell lymphomas is shown, including the mean fluorescence intensity (MFI) of the FITC channel.

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques: Expressing, Gene Expression, Fluorescence, Staining, Control, Negative Control

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: In vitro culture of primary dog DLBCL cells with KtCD40L. (A) Photomicrographs of DLBCL cells alone (left), DLBCL cells cultured with KtCD40L (middle), and KtCD40L alone (right), each shown after seven days in culture. Bars = 100 μm (B) Flow analysis of DLBCL cells before and after culture with KtCD40L for 7 days. Estimates of cell size and complexity were obtained from flow cytometric light scatter properties (upper panels). Viability was determined by 7-AAD exclusion (lower panels). FSC; forward scatter, SSC; side scatter (C) Growth curves of primary dog DLBCL cells (n = 4) cultured with KtCD40L. DLBCL cell numbers were determined at each time point using hemacytometer with dead cell exclusion by trypan blue staining.

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques: In Vitro, Cell Culture, Staining

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: Phenotypic characteristics of DLBCL cells cultured with KtCD40L. (A) DLBCL cells cultured with KtCD40L for 48 days were phenotyped using flow cytometry. Gray lines represent unstained samples. One representative sample of three DLBCL cases analyzed is shown. (B) Clonal IgH gene rearrangements of the same size (105 bp) were observed in tumor cells before (Day 0) and after (Day 48) the culture. One representative sample of two DLBCL cases analyzed is shown. (C) Examples of FISH images obtained from one DLBCL case with trisomy of CFA 31. Control dog metaphase chromosomes (a) and an interphase nucleus (b) probed with a marker for CFA 31 (yellow signal), exhibiting the expected chromosomal location and normal copy number status (n = 2) of this region. Three copies of this locus were evident in 51% of DLBCL cells prior to culture (c), and in 48% of DLBCL cells after culture with KtCD40L for 40 days (d). A KtCD40L cell (upper left without a probe signal) and a DLBCL cell (lower right with probe signals) are shown together in (d).

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques: Cell Culture, Flow Cytometry, Control, Marker

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: Feeder cell-free culture of primary DLBCL cells with recombinant shuCD40L. (A) The MTS cell proliferation assay was performed at 24-hour intervals on primary dog DLBCL cells stimulated with increasing amounts of shuCD40L. Data from 4 independent primary dog DLBCL samples are shown. Data were normalized to results from 5 × 104 cells (0 hour) to show fold increase of cell numbers during the culture period. (B) MTS cell proliferation assay was performed on primary DLBCL cells stimulated with 100 ng/mL shuCD40L in the presence of increasing amounts of anti-human CD40L-IgA. Three different primary DLBCL samples were tested for 72 hours in duplicate conditions and data were normalized to results of cells cultured with 100 ng/mL shuCD40L without neutralizing antibodies. Data shown represent the mean ± SD (n = 3) and statistical significance was tested against the condition of 100 ng/mL shuCD40L without neutralizing antibodies using ANOVA (*P < 0.01, **P < 0.001). (C) Human B-ALL samples were stained for CD22 and CD40. CD22+ tumor cells (> 90% CD22+ cells in both samples) were gated and CD40 expression was analyzed. Gray lines represent staining with an isotype control antibody. (D) The MTS cell proliferation assay using primary human B-ALL cells was performed as described above. Data from 2 independent samples are shown.

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques: Recombinant, Proliferation Assay, Cell Culture, Staining, Expressing, Control

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: IC 50 of 22KDEL and Bic3 for tumor cells

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques:

Journal: Leukemia & lymphoma

Article Title: CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

doi: 10.3109/10428194.2011.654337

Figure Lengend Snippet: Comparable assessment of cytotoxicity using dog and human B-cell malignancies. (A) Four independent primary dog DLBCL samples; #1–3 are the same as in Figure 4, #4 is from a different dog, and (B) two human primary B-ALL samples from different patients than those shown in Figure 4, were cultured for 72 hours with a log serial dilutions (0.01–100 nM) of 22KDEL and Bic3 immunotoxins in the presence of shuCD40L. Experiments were performed in duplicate and cell numbers were determined using the MTS assay. Results with 10 nM immunotoxins are shown, and a summary result of the IC50 for 22KDEL and Bic3 is shown in Table I.

Article Snippet: The human DLBCL cell line SU-DHL4 was obtained from Dr. Lee Honigberg (Pharmacyclics Inc., Sunnyvale, CA).

Techniques: Cell Culture, MTS Assay