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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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More than one ICE homologue is cleaved in cells exposed to an apoptotic stimulus. 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). (A) The time course of induction of apoptosis was determined by flow cytometry as described in Materials and Methods. (B) The time course of cleavage of the proforms of CPP32 (upper panel) and Ich-1 (lower  panel) was determined by Western blot analysis as described in  Materials and Methods. The 32-kD pro-CPP32 is indicated by the  upper arrowhead, and the 17-kD cleavage product is indicated by  the lower arrowhead in the upper panel. The 48-kD pro–Ich-1 is  indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead in the lower panel.
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Figure 1: More than one ICE homologue is cleaved in cells exposed to an apoptotic stimulus. 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). (A) The time course of induction of apoptosis was determined by flow cytometry as described in Materials and Methods. (B) The time course of cleavage of the proforms of CPP32 (upper panel) and Ich-1 (lower panel) was determined by Western blot analysis as described in Materials and Methods. The 32-kD pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead in the upper panel. The 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead in the lower panel.

Mentions: Exposure of THP.1 cells to a number of stimuli, including etoposide, a DNA topoisomerase II inhibitor, or cotreatment with the protein synthesis inhibitor cycloheximide and TLCK, an inhibitor of trypsin-like proteases, induces apoptosis (Zhu et al., 1995). THP.1 cells were coincubated with cycloheximide (25 μM) and TLCK (100 μM) for up to 4 h, and the amount of apoptosis was quantified by flow cytometry (Fig. 1 A). A time-dependent increase in apoptosis was observed, which was first detected at 2 h (Fig. 1 A). To determine whether a time-dependent processing of ICE-like proteases occurred, Western blot analysis was performed using antibodies to the p17 fragment of CPP32 and the p12 fragment of Ich-1. In untreated cells, immunoblots showed the presence of the 32-kD precursor of CPP32 and the 48-kD precursor of Ich-1 (Fig. 1 B, lane 1). After induction of apoptosis, the p17 subunit of the mature CPP32 enzyme was first detected by 2 h and remained elevated until 4 h (Fig. 1 B, lanes 3–5). In parallel, a timedependent decrease in the level of the 32-kD precursor of CPP32 was observed. A time-dependent decrease in the 48-kD precursor protein of Ich-1 accompanied by the formation of its p12 subunit was also observed (Fig. 1 B, lanes 2–5). A small amount of the p12 fragment of Ich-1 was already evident at 1 h (Fig. 1 B, lane 2). Similar results were obtained when apoptosis was induced by etoposide (data not shown).


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)

More than one ICE homologue is cleaved in cells exposed to an apoptotic stimulus. 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). (A) The time course of induction of apoptosis was determined by flow cytometry as described in Materials and Methods. (B) The time course of cleavage of the proforms of CPP32 (upper panel) and Ich-1 (lower  panel) was determined by Western blot analysis as described in  Materials and Methods. The 32-kD pro-CPP32 is indicated by the  upper arrowhead, and the 17-kD cleavage product is indicated by  the lower arrowhead in the upper panel. The 48-kD pro–Ich-1 is  indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead in the lower panel.
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Related In: Results  -  Collection

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Figure 1: More than one ICE homologue is cleaved in cells exposed to an apoptotic stimulus. 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). (A) The time course of induction of apoptosis was determined by flow cytometry as described in Materials and Methods. (B) The time course of cleavage of the proforms of CPP32 (upper panel) and Ich-1 (lower panel) was determined by Western blot analysis as described in Materials and Methods. The 32-kD pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead in the upper panel. The 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead in the lower panel.
Mentions: Exposure of THP.1 cells to a number of stimuli, including etoposide, a DNA topoisomerase II inhibitor, or cotreatment with the protein synthesis inhibitor cycloheximide and TLCK, an inhibitor of trypsin-like proteases, induces apoptosis (Zhu et al., 1995). THP.1 cells were coincubated with cycloheximide (25 μM) and TLCK (100 μM) for up to 4 h, and the amount of apoptosis was quantified by flow cytometry (Fig. 1 A). A time-dependent increase in apoptosis was observed, which was first detected at 2 h (Fig. 1 A). To determine whether a time-dependent processing of ICE-like proteases occurred, Western blot analysis was performed using antibodies to the p17 fragment of CPP32 and the p12 fragment of Ich-1. In untreated cells, immunoblots showed the presence of the 32-kD precursor of CPP32 and the 48-kD precursor of Ich-1 (Fig. 1 B, lane 1). After induction of apoptosis, the p17 subunit of the mature CPP32 enzyme was first detected by 2 h and remained elevated until 4 h (Fig. 1 B, lanes 3–5). In parallel, a timedependent decrease in the level of the 32-kD precursor of CPP32 was observed. A time-dependent decrease in the 48-kD precursor protein of Ich-1 accompanied by the formation of its p12 subunit was also observed (Fig. 1 B, lanes 2–5). A small amount of the p12 fragment of Ich-1 was already evident at 1 h (Fig. 1 B, lane 2). Similar results were obtained when apoptosis was induced by etoposide (data not shown).

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.

Show MeSH
Related in: MedlinePlus