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Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

Alam A, Cohen LY, Aouad S, Sékaly RP - J. Exp. Med. (1999)

Bottom Line: Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate.The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme.Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

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Caspase activation results in selective substrate cleavage. Total proteins from 106 unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 106 Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.
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Figure 6: Caspase activation results in selective substrate cleavage. Total proteins from 106 unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 106 Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.

Mentions: Since several downstream caspases were activated after TCR triggering, we next determined whether caspase substrates were processed in stimulated lymphocytes. PARP was the first caspase-3 substrate identified during apoptosis 29. Stimulation with anti-CD3 and IL-2 increased PARP protein levels within 24 h (Fig. 6), confirming previous reports showing an upregulation of PARP message and activity after PHA stimulation of primary T lymphocytes 4142. Concomitant with its induction, >90% of PARP protein was found cleaved in activated cell lysates (Fig. 6, and see Fig. 8). PARP fragment comigrated in the same gel with the 85-kD form found in apoptotic Jurkat cell extracts, further confirming that caspase activity was induced after primary T cell activation. Another recently identified caspase substrate, the Cdc2 tyrosine kinase Wee1 13, was also upregulated in stimulated PBMCs (Fig. 6). Interestingly, the 97-kD full-length Wee1 that appeared 48 h after stimulation was always detected along with 65-, 34-, and 32-kD bands that result from caspase-mediated cleavage during Fas-mediated apoptosis in Jurkat cells (Fig. 6; reference 13). Therefore, two caspase substrates, PARP and Wee1, were processed in activated T cells in a pattern expected from their cleavage by caspases.


Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

Alam A, Cohen LY, Aouad S, Sékaly RP - J. Exp. Med. (1999)

Caspase activation results in selective substrate cleavage. Total proteins from 106 unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 106 Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.
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Related In: Results  -  Collection

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Figure 6: Caspase activation results in selective substrate cleavage. Total proteins from 106 unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 106 Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.
Mentions: Since several downstream caspases were activated after TCR triggering, we next determined whether caspase substrates were processed in stimulated lymphocytes. PARP was the first caspase-3 substrate identified during apoptosis 29. Stimulation with anti-CD3 and IL-2 increased PARP protein levels within 24 h (Fig. 6), confirming previous reports showing an upregulation of PARP message and activity after PHA stimulation of primary T lymphocytes 4142. Concomitant with its induction, >90% of PARP protein was found cleaved in activated cell lysates (Fig. 6, and see Fig. 8). PARP fragment comigrated in the same gel with the 85-kD form found in apoptotic Jurkat cell extracts, further confirming that caspase activity was induced after primary T cell activation. Another recently identified caspase substrate, the Cdc2 tyrosine kinase Wee1 13, was also upregulated in stimulated PBMCs (Fig. 6). Interestingly, the 97-kD full-length Wee1 that appeared 48 h after stimulation was always detected along with 65-, 34-, and 32-kD bands that result from caspase-mediated cleavage during Fas-mediated apoptosis in Jurkat cells (Fig. 6; reference 13). Therefore, two caspase substrates, PARP and Wee1, were processed in activated T cells in a pattern expected from their cleavage by caspases.

Bottom Line: Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate.The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme.Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

Show MeSH
Related in: MedlinePlus