Figure 1.
Figure 1. Cross-linked Hu1D10 induces apoptosis in CLL cells. (A) CLL samples were treated for 4 hours with either (i) Hu1D10 (10 μg/mL), (ii) Hu1D10 (10μg/mL) with media containing 30% autologous serum, (iii) trastuzumab plus antihuman Fc antibody (both 10 μg/mL), or (iv) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL). Following annexin V–FITC/PI staining, samples were analyzed by flow cytometry. Values represent the percentage of positive cells above that measured for untreated cells. Error bars indicate the SD among the different samples and thus represent heterogeneity of the samples rather than measurement error. All samples strongly expressed the 1D10 antigen. (B) A representative annexin V–FITC/PI dot plot after media (i) or Hu1D10 plus antihuman Fc antibody (iv). The numbers in each quadrant indicate the percent of cells labeled with annexin V–FITC (lower right), PI (upper left), or annexin V–FITC and PI (upper right). (C) After a 4-hour incubation without (i) or with (iv) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL), cells were incubated with 50 ng/mL rhodamine 123 and then analyzed by flow cytometry. The rise in the lower intensity peak indicated by the arrow, with antibody treatment, indicates loss of the fluorescent dye rhodamine 123 from mitochondria. Histograms in panel C are representative of experiments performed on 4 different CLL patient samples. (D) Protein lysates from cells incubated for 4 hours in media (lanes 3 and 6), trastuzumab plus antihuman Fc antibody (both 10 μg/m; lanes 4 and 7) or Hu1D10 plus antihuman Fc antibody (both 10 μg/mL; lanes 5 and 8) were probed for PARP and caspases 3, 8, and 9. Unlike UV-irradiated Jurkat cell–positive control (lane 2), no processing of the caspases or PARP was observed despite apoptosis as depicted in panels A-C. (E) Prior to treating cells with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, cells were incubated for 30 minutes with (iii) or without (ii) cytochalasin B (20 μM). Cells treated with cytochalasin B only are indicated by I. Following treatment, cells were stained with annexin V–FITC/PI and analyzed by flow cytometry. Values shown are percent positive cells above untreated samples. Error bars indicate the SD among 4 different CLL samples tested. (F) Cells were incubated for 30 minutes in media (i-ii) or media plus 10 mM methyl-B-cyclodextran (iii-iv). Samples represented by ii and iv were then treated with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours. Cells were then stained with annexin V–FITC/PI and analyzed by flow cytometry. Note that the relative difference between the methyl-B-cyclodextran–treated samples (13.8%) is significantly less than the untreated samples (26.2%).

Cross-linked Hu1D10 induces apoptosis in CLL cells. (A) CLL samples were treated for 4 hours with either (i) Hu1D10 (10 μg/mL), (ii) Hu1D10 (10μg/mL) with media containing 30% autologous serum, (iii) trastuzumab plus antihuman Fc antibody (both 10 μg/mL), or (iv) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL). Following annexin V–FITC/PI staining, samples were analyzed by flow cytometry. Values represent the percentage of positive cells above that measured for untreated cells. Error bars indicate the SD among the different samples and thus represent heterogeneity of the samples rather than measurement error. All samples strongly expressed the 1D10 antigen. (B) A representative annexin V–FITC/PI dot plot after media (i) or Hu1D10 plus antihuman Fc antibody (iv). The numbers in each quadrant indicate the percent of cells labeled with annexin V–FITC (lower right), PI (upper left), or annexin V–FITC and PI (upper right). (C) After a 4-hour incubation without (i) or with (iv) Hu1D10 plus antihuman Fc antibody (both 10 μg/mL), cells were incubated with 50 ng/mL rhodamine 123 and then analyzed by flow cytometry. The rise in the lower intensity peak indicated by the arrow, with antibody treatment, indicates loss of the fluorescent dye rhodamine 123 from mitochondria. Histograms in panel C are representative of experiments performed on 4 different CLL patient samples. (D) Protein lysates from cells incubated for 4 hours in media (lanes 3 and 6), trastuzumab plus antihuman Fc antibody (both 10 μg/m; lanes 4 and 7) or Hu1D10 plus antihuman Fc antibody (both 10 μg/mL; lanes 5 and 8) were probed for PARP and caspases 3, 8, and 9. Unlike UV-irradiated Jurkat cell–positive control (lane 2), no processing of the caspases or PARP was observed despite apoptosis as depicted in panels A-C. (E) Prior to treating cells with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours, cells were incubated for 30 minutes with (iii) or without (ii) cytochalasin B (20 μM). Cells treated with cytochalasin B only are indicated by I. Following treatment, cells were stained with annexin V–FITC/PI and analyzed by flow cytometry. Values shown are percent positive cells above untreated samples. Error bars indicate the SD among 4 different CLL samples tested. (F) Cells were incubated for 30 minutes in media (i-ii) or media plus 10 mM methyl-B-cyclodextran (iii-iv). Samples represented by ii and iv were then treated with Hu1D10 plus antihuman Fc antibody (both 10 μg/mL) for 4 hours. Cells were then stained with annexin V–FITC/PI and analyzed by flow cytometry. Note that the relative difference between the methyl-B-cyclodextran–treated samples (13.8%) is significantly less than the untreated samples (26.2%).

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