Figure 1
Figure 1. Direct and indirect induction of cell death by rituximab in B-NHL cell lines. (A) Human B-NHL cell lines were incubated with monomeric rituximab (R), rituximab cross-linked by an anti-Ig F(ab′)2 fragment (Rc), the anti-Ig F(ab′)2 fragment alone (F(ab′)2), or the isotype control for 48 hours. The CD20-negative human leukemia cell line K562 served as negative control. Cell death was quantified flow cytometrically after staining with PI; mean values plus SD of 3 independent experiments are shown. (B) Human B-NHL cell lines were incubated with cross-linked rituximab (apoptosis), monomeric rituximab, and mononuclear cells (ADCC), or monomeric rituximab and human serum (CDC). The CD20-negative human leukemia cell line K562 served as negative control. Cell death was quantified flow cytometrically after staining with PI; mean values plus SD of 3 independent experiments are shown. (C) The 3 rituximab-sensitive B-NHL cell lines (SU-DHL-4, Ramos, WSU-NHL) were incubated with cross-linked rituximab in the presence of vehicle (Rc) or the broad-spectrum caspase inhibitor zVAD-fmk (50 μM, Rc + zVAD) for 48 hours. Cells with apoptotic DNA fragmentation were quantified flow cytometrically after hypotonic lysis and staining with PI. Mean values plus SD of 3 independent experiments are given (left panel). The same B-NHL cell lines were incubated with cross-linked rituximab (Rc) for 24 hours, and the fraction of cells with caspase-3-like activity was determined flow cytometrically after staining with the fluorescent caspase substrate FITC-VAD. Mean values plus SD of 3 independent experiments are shown (right panel). (D) Rituximab-sensitive Ramos B-NHL cells and rituximab-resistant Jeko-1 B-NHL cells were incubated with monomeric (R) or cross-linked rituximab (Rc) for 24 hours. The fraction of cells with dissipated mitochondrial transmembrane potential Δψm was determined flow cytometrically by loss of staining with the fluorescent mitochondrial dye TMRE. Representative histograms of at least 3 independent repeat experiments are shown. Note the loss of TMRE staining in Ramos, but not in Jeko-1 cells after treatment with cross-linked rituximab.

Direct and indirect induction of cell death by rituximab in B-NHL cell lines. (A) Human B-NHL cell lines were incubated with monomeric rituximab (R), rituximab cross-linked by an anti-Ig F(ab′)2 fragment (Rc), the anti-Ig F(ab′)2 fragment alone (F(ab′)2), or the isotype control for 48 hours. The CD20-negative human leukemia cell line K562 served as negative control. Cell death was quantified flow cytometrically after staining with PI; mean values plus SD of 3 independent experiments are shown. (B) Human B-NHL cell lines were incubated with cross-linked rituximab (apoptosis), monomeric rituximab, and mononuclear cells (ADCC), or monomeric rituximab and human serum (CDC). The CD20-negative human leukemia cell line K562 served as negative control. Cell death was quantified flow cytometrically after staining with PI; mean values plus SD of 3 independent experiments are shown. (C) The 3 rituximab-sensitive B-NHL cell lines (SU-DHL-4, Ramos, WSU-NHL) were incubated with cross-linked rituximab in the presence of vehicle (Rc) or the broad-spectrum caspase inhibitor zVAD-fmk (50 μM, Rc + zVAD) for 48 hours. Cells with apoptotic DNA fragmentation were quantified flow cytometrically after hypotonic lysis and staining with PI. Mean values plus SD of 3 independent experiments are given (left panel). The same B-NHL cell lines were incubated with cross-linked rituximab (Rc) for 24 hours, and the fraction of cells with caspase-3-like activity was determined flow cytometrically after staining with the fluorescent caspase substrate FITC-VAD. Mean values plus SD of 3 independent experiments are shown (right panel). (D) Rituximab-sensitive Ramos B-NHL cells and rituximab-resistant Jeko-1 B-NHL cells were incubated with monomeric (R) or cross-linked rituximab (Rc) for 24 hours. The fraction of cells with dissipated mitochondrial transmembrane potential Δψm was determined flow cytometrically by loss of staining with the fluorescent mitochondrial dye TMRE. Representative histograms of at least 3 independent repeat experiments are shown. Note the loss of TMRE staining in Ramos, but not in Jeko-1 cells after treatment with cross-linked rituximab.

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