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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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YVAD.CMK inhibits cleavage of lamins A/B but not of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone  (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3). (A) Cell  samples were analyzed by Western blot analysis using antibodies to CPP32 and Ich-1 as described in Materials and Methods. (B) Cells  were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of the  indicated concentrations of YVAD.CMK (5–25 μM) (lanes 3–5). The cleavage of intact lamin A/B (66 kD) to a fragment of 46 kD was  detected by Western blot analysis using a lamin A/B antibody. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of Z-VAD.FMK (50 μM) (lane 3) or YVAD.CMK (25 μM)  (lane 4). Cell samples were analyzed by Western blot analysis using an antibody to Mch2α.
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Figure 4: YVAD.CMK inhibits cleavage of lamins A/B but not of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3). (A) Cell samples were analyzed by Western blot analysis using antibodies to CPP32 and Ich-1 as described in Materials and Methods. (B) Cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of the indicated concentrations of YVAD.CMK (5–25 μM) (lanes 3–5). The cleavage of intact lamin A/B (66 kD) to a fragment of 46 kD was detected by Western blot analysis using a lamin A/B antibody. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of Z-VAD.FMK (50 μM) (lane 3) or YVAD.CMK (25 μM) (lane 4). Cell samples were analyzed by Western blot analysis using an antibody to Mch2α.

Mentions: THP.1 cells were preincubated for 1 h either alone or with YVAD.CMK (5–25 μM), and then further incubated for 4 h with cycloheximide (25 μM) and TLCK (100 μM). Apoptosis was assessed by flow cytometry. Cycloheximide in the presence of TLCK induced ∼48% apoptosis that was not affected by pretreatment with YVAD.CMK (∼46%). Thus, YVAD.CMK did not inhibit apoptosis in THP.1 cells, assessed either by flow cytometry or by internucleosomal cleavage of DNA (Zhu et al., 1995). Furthermore, no significant inhibition of the processing of pro-CPP32 or pro– Ich-1 was observed in cells pretreated with YVAD.CMK (25 μM) (Fig. 4 A). The processed fragment of CPP32 detected in the presence of YVAD.CMK (Fig. 4 A, lane 3) was slightly larger and did not comigrate with the fragments obtained after treatment with the apoptotic stimulus alone (Fig. 4 A, lane 2). This suggested that, while YVAD.CMK did not significantly inhibit the loss of proCPP32, it did inhibit further degradation of the initial cleaved product. In contrast to its inability to inhibit processing of CPP32 and Ich-1, YVAD.CMK was a very good inhibitor of the cleavage of lamins A/B (Fig. 4 B). Control cells contained only intact 66-kD lamin A/B (Fig. 4 B, lane 1), which was cleaved to a 46-kD fragment in the presence of cycloheximide and TLCK (Fig. 4 B, lane 2). This cleavage was almost totally inhibited by YVAD.CMK (5–25 μM) (Fig. 4 B, lanes 3–5). Essentially similar results were obtained with etoposide (data not shown). In contrast with the observed inhibition of lamin cleavage, these concentrations of YVAD.CMK did not inhibit PARP cleavage (Browne, S., M. MacFarlane, G.M. Cohen, and C. Paraskeva, manuscript submitted for publication).


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)

YVAD.CMK inhibits cleavage of lamins A/B but not of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone  (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3). (A) Cell  samples were analyzed by Western blot analysis using antibodies to CPP32 and Ich-1 as described in Materials and Methods. (B) Cells  were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of the  indicated concentrations of YVAD.CMK (5–25 μM) (lanes 3–5). The cleavage of intact lamin A/B (66 kD) to a fragment of 46 kD was  detected by Western blot analysis using a lamin A/B antibody. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of Z-VAD.FMK (50 μM) (lane 3) or YVAD.CMK (25 μM)  (lane 4). Cell samples were analyzed by Western blot analysis using an antibody to Mch2α.
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Figure 4: YVAD.CMK inhibits cleavage of lamins A/B but not of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3). (A) Cell samples were analyzed by Western blot analysis using antibodies to CPP32 and Ich-1 as described in Materials and Methods. (B) Cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of the indicated concentrations of YVAD.CMK (5–25 μM) (lanes 3–5). The cleavage of intact lamin A/B (66 kD) to a fragment of 46 kD was detected by Western blot analysis using a lamin A/B antibody. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of Z-VAD.FMK (50 μM) (lane 3) or YVAD.CMK (25 μM) (lane 4). Cell samples were analyzed by Western blot analysis using an antibody to Mch2α.
Mentions: THP.1 cells were preincubated for 1 h either alone or with YVAD.CMK (5–25 μM), and then further incubated for 4 h with cycloheximide (25 μM) and TLCK (100 μM). Apoptosis was assessed by flow cytometry. Cycloheximide in the presence of TLCK induced ∼48% apoptosis that was not affected by pretreatment with YVAD.CMK (∼46%). Thus, YVAD.CMK did not inhibit apoptosis in THP.1 cells, assessed either by flow cytometry or by internucleosomal cleavage of DNA (Zhu et al., 1995). Furthermore, no significant inhibition of the processing of pro-CPP32 or pro– Ich-1 was observed in cells pretreated with YVAD.CMK (25 μM) (Fig. 4 A). The processed fragment of CPP32 detected in the presence of YVAD.CMK (Fig. 4 A, lane 3) was slightly larger and did not comigrate with the fragments obtained after treatment with the apoptotic stimulus alone (Fig. 4 A, lane 2). This suggested that, while YVAD.CMK did not significantly inhibit the loss of proCPP32, it did inhibit further degradation of the initial cleaved product. In contrast to its inability to inhibit processing of CPP32 and Ich-1, YVAD.CMK was a very good inhibitor of the cleavage of lamins A/B (Fig. 4 B). Control cells contained only intact 66-kD lamin A/B (Fig. 4 B, lane 1), which was cleaved to a 46-kD fragment in the presence of cycloheximide and TLCK (Fig. 4 B, lane 2). This cleavage was almost totally inhibited by YVAD.CMK (5–25 μM) (Fig. 4 B, lanes 3–5). Essentially similar results were obtained with etoposide (data not shown). In contrast with the observed inhibition of lamin cleavage, these concentrations of YVAD.CMK did not inhibit PARP cleavage (Browne, S., M. MacFarlane, G.M. Cohen, and C. Paraskeva, manuscript submitted for publication).

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