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

Kashkar H, Haefs C, Shin H, Hamilton-Dutoit SJ, Salvesen GS, Kronke M, Jurgensmeier JM - J. Exp. Med. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19-21, 50935 Cologne, Germany.

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.

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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.
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fig5: 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.

Mentions: If XIAP was the key inhibitor of apoptosis in HL-derived B cells, removal of XIAP should relieve the inhibition of caspase-3 activation. Indeed, in the presence of Smac, a mitochondrial inhibitor of XIAP, the ability of cytochrome c was restored to trigger processing and activation of caspase-3 in HL-derived B cells (Fig. 5, A and B)Figure 5.


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

Kashkar H, Haefs C, Shin H, Hamilton-Dutoit SJ, Salvesen GS, Kronke M, Jurgensmeier JM - J. Exp. Med. (2003)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194071&req=5

fig5: 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.
Mentions: If XIAP was the key inhibitor of apoptosis in HL-derived B cells, removal of XIAP should relieve the inhibition of caspase-3 activation. Indeed, in the presence of Smac, a mitochondrial inhibitor of XIAP, the ability of cytochrome c was restored to trigger processing and activation of caspase-3 in HL-derived B cells (Fig. 5, A and B)Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19-21, 50935 Cologne, Germany.

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.

Show MeSH
Related in: MedlinePlus