XIAP-mediated caspase inhibition in Hodgkin's lymphoma-derived B cells

Abstract

The malignant Hodgkin and Reed-Sternberg cells of Hodgkin's lymphoma (HL) and HL-derived B cell lines were previously shown to be resistant to different apoptotic stimuli. We show here that cytochrome c fails to stimulate caspases-9 and -3 activation in cytosolic extracts of HL-derived B cells, which is due to high level expression of X-linked inhibitor of apoptosis (XIAP). Coimmunoprecipitation studies revealed that XIAP, apoptosis protease-activating factor-1, and caspase-3 are complexed in HL-derived B cell lysates. Even after stimulation with exogenous cytochrome c and dATP, XIAP impairs the proteolytic processing and activation of caspase-3. In cytosolic extracts, inhibition of XIAP by the second mitochondria-derived activator of caspases (Smac)/DIABLO, or immunodepletion of XIAP restores cytochrome c-triggered processing and activation of caspase-3. Smac or a Smac-derived agonistic peptide also sensitized intact HL-derived B cells for the apoptotic action of staurosporine. Finally, Hodgkin and Reed-Sternberg cells of primary tumor HL tissues also constitutively and abundantly express XIAP. The results of this paper suggest that high level XIAP expression is a hallmark of HL, which may play a crucial role in resistance to apoptosis.

Figure 1.

Failure of cytochrome c to induce caspase activation in cytosolic extracts of HL-derived B cell lines. Cytosolic extracts of L1236, L591, L428, and KMH2 cells, and of control B cell L1309 were prepared and equal amounts of protein were incubated with or without cytochrome c/dATP for 1 h at 30°C. Cytosolic extracts were resolved by SDS-PAGE and subjected to Western blot analysis. (A) Procaspase-9 (p46) and its fragments (p37, p35) were detected by polyclonal rabbit anti–caspase-9 antibody. (B) Procaspase-3 (p32) and its fragments (p20, p17) were detected by polyclonal anti–caspase-3 antibody. (C) Measurement of relative caspase activity using DEVD-AFC. Samples were normalized for total cytosolic protein content. Asterisk indicates nonspecific bands recognized by polyclonal antibodies.

Figure 2.

Expression of caspase activators and inhibitors in HL-derived B cell lines. (A) Equal amounts of proteins from total cell lysate of L1236, L591, L428, and KMH2 cells and of control B cells L1309 were subjected to SDS-PAGE and Western blot analysis. Proteins were detected using antibodies against Apaf-1 and XIAP. (B) XIAP expression in primary Hodgkin's lymphoma (HL) tissues. XIAP positivity in H-RS cells: + = very weak; ++ = weak; +++ = moderate; ++++ = strong staining. (C) Paraffin section immunohistochemistry of cases 3, 5, 6, 7, 10, and 11 of classical HL cases listed in Fig. 2 B. XIAP was stained using anti-XIAP specific mAb. There are strong (Nos. 5, 6, 7, and 11) or moderate (Nos. 3 and 10) granular intracytoplasmic staining for XIAP in essentially all morphologically recognizable Hodgkin or Reed-Sternberg cells. Background lymphocytes are negative for XIAP staining.

Figure 2.

Expression of caspase activators and inhibitors in HL-derived B cell lines. (A) Equal amounts of proteins from total cell lysate of L1236, L591, L428, and KMH2 cells and of control B cells L1309 were subjected to SDS-PAGE and Western blot analysis. Proteins were detected using antibodies against Apaf-1 and XIAP. (B) XIAP expression in primary Hodgkin's lymphoma (HL) tissues. XIAP positivity in H-RS cells: + = very weak; ++ = weak; +++ = moderate; ++++ = strong staining. (C) Paraffin section immunohistochemistry of cases 3, 5, 6, 7, 10, and 11 of classical HL cases listed in Fig. 2 B. XIAP was stained using anti-XIAP specific mAb. There are strong (Nos. 5, 6, 7, and 11) or moderate (Nos. 3 and 10) granular intracytoplasmic staining for XIAP in essentially all morphologically recognizable Hodgkin or Reed-Sternberg cells. Background lymphocytes are negative for XIAP staining.

Figure 2.

Expression of caspase activators and inhibitors in HL-derived B cell lines. (A) Equal amounts of proteins from total cell lysate of L1236, L591, L428, and KMH2 cells and of control B cells L1309 were subjected to SDS-PAGE and Western blot analysis. Proteins were detected using antibodies against Apaf-1 and XIAP. (B) XIAP expression in primary Hodgkin's lymphoma (HL) tissues. XIAP positivity in H-RS cells: + = very weak; ++ = weak; +++ = moderate; ++++ = strong staining. (C) Paraffin section immunohistochemistry of cases 3, 5, 6, 7, 10, and 11 of classical HL cases listed in Fig. 2 B. XIAP was stained using anti-XIAP specific mAb. There are strong (Nos. 5, 6, 7, and 11) or moderate (Nos. 3 and 10) granular intracytoplasmic staining for XIAP in essentially all morphologically recognizable Hodgkin or Reed-Sternberg cells. Background lymphocytes are negative for XIAP staining.

Figure 3.

XIAP coimmunoprecipitates with activated caspase-3. Cytosolic extracts of L1236, L591, L428, and KMH2 cells and of control B cells L1309 were prepared, and equal amounts of protein were incubated with or without cytochrome c/dATP for 1 h at 30°C. (A) 100 μg samples were resolved by SDS-PAGE and subjected to Western blot analysis. XIAP was detected by mouse anti-XIAP mAb. (B–D) Caspase-3, Apaf-1, and Bax were immunoprecipitated by mouse anti–caspase-3, rat anti–Apaf-1, and mouse anti-Bax mAb in 600-μg cytosolic extracts and subjected to SDS-PAGE and Western blotting. XIAP, caspase-3, Apaf-1, and Bax were detected by mouse anti-XIAP mAb, polyclonal rabbit anti–caspase-3, polyclonal rabbit anti–Apaf-1, and polyclonal rabbit anti-Bax antibodies. Asterisk indicates mouse IgG.

Figure 4.

Caspase-3 activation by caspase-8 and granzyme-B. Cytosolic extracts of L1236, L591, L428, and KMH2 cells and of control B cells L1309 were prepared, and equal amounts of protein were left untreated or incubated for 1 h with recombinant active caspase-8 (A) and granzyme-B (B) at 30°C. Cytosolic extracts were resolved by SDS-PAGE and subjected to Western blot analysis. Caspase-3 and XIAP were detected by polyclonal rabbit anti–caspase-3 antibody and mouse anti-XIAP mAb. Relative caspase activity was measured using DEVD-AFC after incubation of cytosolic extracts with recombinant active caspase-8 (A)and granzyme-B (B). Samples were normalized for total cytosolic protein content.

Figure 5.

Depletion of XIAP restores caspase-3 processing and activity. Cytosolic extracts of L1236 and KMH2 cells and of control B cell L1309 were prepared, and equal amounts of protein were incubated with or without cytochrome c/dATP in the absence and presence of Smac protein for 1 h at 30°C. (A) Cytosolic extracts were resolved by SDS-PAGE and subjected to Western blot analysis. Caspase-3 was detected by polyclonal rabbit anti–caspase-3 antibody. (B) Relative caspase activity was measured by hydrolysis of DEVD-AFC. Samples were normalized for total cytosolic protein content. (C) XIAP was immunodepleted by mouse anti-XIAP mAb. Cytosolic extracts of KMH2 cells with or without XIAP were incubated with or without cytochrome c/dATP for 1 h at 30°C. Samples were normalized for total cytosolic protein content and relative caspase-3 activity was measured by DEVDase activity. (D) Cells were transfected with vehicle, β-galactosidase, Smac N7 peptide, or Smac protein and treated for 24 h with 1 μM staurosporine. Cell death was determined by trypan blue exclusion. Each value represents the average of results from two independent experiments.

Figure 5.

Depletion of XIAP restores caspase-3 processing and activity. Cytosolic extracts of L1236 and KMH2 cells and of control B cell L1309 were prepared, and equal amounts of protein were incubated with or without cytochrome c/dATP in the absence and presence of Smac protein for 1 h at 30°C. (A) Cytosolic extracts were resolved by SDS-PAGE and subjected to Western blot analysis. Caspase-3 was detected by polyclonal rabbit anti–caspase-3 antibody. (B) Relative caspase activity was measured by hydrolysis of DEVD-AFC. Samples were normalized for total cytosolic protein content. (C) XIAP was immunodepleted by mouse anti-XIAP mAb. Cytosolic extracts of KMH2 cells with or without XIAP were incubated with or without cytochrome c/dATP for 1 h at 30°C. Samples were normalized for total cytosolic protein content and relative caspase-3 activity was measured by DEVDase activity. (D) Cells were transfected with vehicle, β-galactosidase, Smac N7 peptide, or Smac protein and treated for 24 h with 1 μM staurosporine. Cell death was determined by trypan blue exclusion. Each value represents the average of results from two independent experiments.