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Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells.

MacFarlane M, Cain K, Sun XM, Alnemri ES, Cohen GM - J. Cell Biol. (1997)

Bottom Line: These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha.Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells.This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Mechanisms of Human Toxicity, University of Leicester, United Kingdom.

ABSTRACT
Identification of the processing/activation of multiple interleukin-1beta converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3alpha but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3alpha. was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3alpha or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3alpha, Mch2alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.

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Processing of Mch3α in apoptotic THP.1 cells: inhibition by ZVAD.FMK but not by YVAD.CMK. (A) THP.1 cells  were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM).  Where indicated, cells were pretreated for 1 h with Z-VAD.FMK  (50 μM), and then incubated for 4 h in the presence of the apoptotic stimulus. The time course of processing of the proform of  Mch3α (lanes 1–5) and its inhibition by Z-VAD.FMK (lane 6)  was determined by Western blot analysis as described in Materials and Methods. (B) THP.1 cells were incubated for 4 h in the  presence of etoposide (25 μM), stained with Hoechst 33342 and  propidium iodide, and then sorted by flow cytometry as previously described (see Materials and Methods and Fig. 2). Cells  with either normal (lane 1) or apoptotic (lane 2) morphology  were analyzed by Western blot analysis. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (25 μM)  and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK  (25 μM) (lane 3), and samples were analyzed by Western blot  analysis. The proforms of Mch3α and its cleavage product are indicated by the upper and lower arrowheads, respectively (A–C).
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Figure 6: Processing of Mch3α in apoptotic THP.1 cells: inhibition by ZVAD.FMK but not by YVAD.CMK. (A) THP.1 cells were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM). Where indicated, cells were pretreated for 1 h with Z-VAD.FMK (50 μM), and then incubated for 4 h in the presence of the apoptotic stimulus. The time course of processing of the proform of Mch3α (lanes 1–5) and its inhibition by Z-VAD.FMK (lane 6) was determined by Western blot analysis as described in Materials and Methods. (B) THP.1 cells were incubated for 4 h in the presence of etoposide (25 μM), stained with Hoechst 33342 and propidium iodide, and then sorted by flow cytometry as previously described (see Materials and Methods and Fig. 2). Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3), and samples were analyzed by Western blot analysis. The proforms of Mch3α and its cleavage product are indicated by the upper and lower arrowheads, respectively (A–C).

Mentions: Characterization of the proteolytic activity present in lysates suggested the activation of another CPP32-like protease, in addition to CPP32 itself, in the execution phase of apoptosis in THP.1 cells. Kinetic data suggested that this protease was Mch3α, and to verify we raised an antibody to the p17 subunit of Mch3α. THP.1 cells were coincubated with cycloheximide (25 μM) and TLCK (100 μM) for up to 4 h, and the cleavage of Mch3α was assessed (Fig. 6 A). In untreated cells, immunoblots showed the presence of the 34-kD precursor form of Mch3α (Fig. 6 A, lane 1). After induction of apoptosis, the p19 subunit of Mch3α was first detected by 2 h and remained evident until 4 h (Fig. 6 A, lanes 2–5). In parallel, a time-dependent decrease in the 34-kD proform of Mch3α was observed, coincident with the induction of apoptosis as previously assessed by flow cytometry (Fig. 1 A). Similar results were obtained when apoptosis was induced by etoposide (data not shown). To determine whether processing of Mch3α correlated with apoptotic execution, pure populations of normal and apoptotic cells were obtained and analyzed. THP.1 cells were exposed for 4 h to etoposide (25 μM), and cells displaying either normal or apoptotic morphology were separated by fluorescence-activated cell sorting. Cells displaying normal morphology contained only the proform of Mch3α (Fig. 6 B, lane 1), whereas apoptotic cells contained the catalytically active subunit p19 with almost no intact Mch3α (Fig. 6 B, lane 2). Thus, no cleavage of Mch3α was observed until cells displaying normal morphology acquired apoptotic morphology, suggesting that activation of Mch3α had occurred during the execution phase of apoptosis.


Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells.

MacFarlane M, Cain K, Sun XM, Alnemri ES, Cohen GM - J. Cell Biol. (1997)

Processing of Mch3α in apoptotic THP.1 cells: inhibition by ZVAD.FMK but not by YVAD.CMK. (A) THP.1 cells  were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM).  Where indicated, cells were pretreated for 1 h with Z-VAD.FMK  (50 μM), and then incubated for 4 h in the presence of the apoptotic stimulus. The time course of processing of the proform of  Mch3α (lanes 1–5) and its inhibition by Z-VAD.FMK (lane 6)  was determined by Western blot analysis as described in Materials and Methods. (B) THP.1 cells were incubated for 4 h in the  presence of etoposide (25 μM), stained with Hoechst 33342 and  propidium iodide, and then sorted by flow cytometry as previously described (see Materials and Methods and Fig. 2). Cells  with either normal (lane 1) or apoptotic (lane 2) morphology  were analyzed by Western blot analysis. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (25 μM)  and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK  (25 μM) (lane 3), and samples were analyzed by Western blot  analysis. The proforms of Mch3α and its cleavage product are indicated by the upper and lower arrowheads, respectively (A–C).
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Figure 6: Processing of Mch3α in apoptotic THP.1 cells: inhibition by ZVAD.FMK but not by YVAD.CMK. (A) THP.1 cells were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM). Where indicated, cells were pretreated for 1 h with Z-VAD.FMK (50 μM), and then incubated for 4 h in the presence of the apoptotic stimulus. The time course of processing of the proform of Mch3α (lanes 1–5) and its inhibition by Z-VAD.FMK (lane 6) was determined by Western blot analysis as described in Materials and Methods. (B) THP.1 cells were incubated for 4 h in the presence of etoposide (25 μM), stained with Hoechst 33342 and propidium iodide, and then sorted by flow cytometry as previously described (see Materials and Methods and Fig. 2). Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3), and samples were analyzed by Western blot analysis. The proforms of Mch3α and its cleavage product are indicated by the upper and lower arrowheads, respectively (A–C).
Mentions: Characterization of the proteolytic activity present in lysates suggested the activation of another CPP32-like protease, in addition to CPP32 itself, in the execution phase of apoptosis in THP.1 cells. Kinetic data suggested that this protease was Mch3α, and to verify we raised an antibody to the p17 subunit of Mch3α. THP.1 cells were coincubated with cycloheximide (25 μM) and TLCK (100 μM) for up to 4 h, and the cleavage of Mch3α was assessed (Fig. 6 A). In untreated cells, immunoblots showed the presence of the 34-kD precursor form of Mch3α (Fig. 6 A, lane 1). After induction of apoptosis, the p19 subunit of Mch3α was first detected by 2 h and remained evident until 4 h (Fig. 6 A, lanes 2–5). In parallel, a time-dependent decrease in the 34-kD proform of Mch3α was observed, coincident with the induction of apoptosis as previously assessed by flow cytometry (Fig. 1 A). Similar results were obtained when apoptosis was induced by etoposide (data not shown). To determine whether processing of Mch3α correlated with apoptotic execution, pure populations of normal and apoptotic cells were obtained and analyzed. THP.1 cells were exposed for 4 h to etoposide (25 μM), and cells displaying either normal or apoptotic morphology were separated by fluorescence-activated cell sorting. Cells displaying normal morphology contained only the proform of Mch3α (Fig. 6 B, lane 1), whereas apoptotic cells contained the catalytically active subunit p19 with almost no intact Mch3α (Fig. 6 B, lane 2). Thus, no cleavage of Mch3α was observed until cells displaying normal morphology acquired apoptotic morphology, suggesting that activation of Mch3α had occurred during the execution phase of apoptosis.

Bottom Line: These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha.Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells.This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Mechanisms of Human Toxicity, University of Leicester, United Kingdom.

ABSTRACT
Identification of the processing/activation of multiple interleukin-1beta converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3alpha but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3alpha. was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3alpha or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3alpha, Mch2alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.

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Related in: MedlinePlus