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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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Z.DEVD-AFC cleavage activity in lysates derived from  THP.1 cells committed to apoptosis precedes the morphological  characteristics of cell death. THP.1 cells were incubated for up to  4 h, either alone (Con) (○–○) or in the presence of cycloheximide (CHX) and TLCK (•–•; ▪–▪), and lysates were prepared  at the indicated time points as described in Materials and Methods. Z-DEVD.AFC (○–○; •–•) and Ac-YVAD.AMC (▪–▪)  hydrolysis activities were determined as described in Materials  and Methods and are shown in A. The extent of apoptosis at each  time point, as assessed by flow cytometry, is indicated in brackets.  Lysates from both untreated (Con) and THP.1 cells exposed to  an apoptotic stimulus (CHX + TLCK) were analyzed by Western  blot analysis using antibodies to CPP32 as described in Materials and Methods. (B) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower  arrowhead. The time course of cleavage of CPP32 (B) paralleled  the hydrolysis activity of the lysates towards Z-DEVD.AFC.
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Figure 5: Z.DEVD-AFC cleavage activity in lysates derived from THP.1 cells committed to apoptosis precedes the morphological characteristics of cell death. THP.1 cells were incubated for up to 4 h, either alone (Con) (○–○) or in the presence of cycloheximide (CHX) and TLCK (•–•; ▪–▪), and lysates were prepared at the indicated time points as described in Materials and Methods. Z-DEVD.AFC (○–○; •–•) and Ac-YVAD.AMC (▪–▪) hydrolysis activities were determined as described in Materials and Methods and are shown in A. The extent of apoptosis at each time point, as assessed by flow cytometry, is indicated in brackets. Lysates from both untreated (Con) and THP.1 cells exposed to an apoptotic stimulus (CHX + TLCK) were analyzed by Western blot analysis using antibodies to CPP32 as described in Materials and Methods. (B) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead. The time course of cleavage of CPP32 (B) paralleled the hydrolysis activity of the lysates towards Z-DEVD.AFC.

Mentions: The processing/cleavage of CPP32, Ich-1, Mch2α, PARP, U1-70K, and lamins A/B strongly suggested that activation of an ICE-like proteolytic activity was an important effector of the cell death program. This was further investigated using lysates from unsorted control and apoptotic cells to assay for proteolytic activity with Z-DEVD.AFC. This model substrate was chosen because the DEVD tetrapeptide sequence is identical to the cleavage site of PARP, i.e., DEVD216-G217. In addition, CPP32 (Nicholson et al., 1995), Mch2α, and Mch3α (Fernandes-Alnemri et al., 1995a,b) cleave the similar substrate analogue, Ac-DEVD.AMC. The Z-DEVD.AFC cleaving activity of lysates obtained from control cells was very low and did not increase after incubation of cells for 4 h (Fig. 5 A). In contrast, increased proteolytic activity was observed in lysates prepared from cells exposed to an apoptotic stimulus (Fig. 5 A). Within 1 h of exposure to the apoptotic stimulus, a small increase in Z-DEVD.AFC cleavage activity was detected in lysates (Fig. 5 A), which preceded the onset of apoptosis in intact cells, as measured by flow cytometry (Figs. 1 A and 5 A). The maximal increase in proteolytic activity occurred at 2 h and was ∼15-fold greater than the activity in control lysates (Fig. 5 A).


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)

Z.DEVD-AFC cleavage activity in lysates derived from  THP.1 cells committed to apoptosis precedes the morphological  characteristics of cell death. THP.1 cells were incubated for up to  4 h, either alone (Con) (○–○) or in the presence of cycloheximide (CHX) and TLCK (•–•; ▪–▪), and lysates were prepared  at the indicated time points as described in Materials and Methods. Z-DEVD.AFC (○–○; •–•) and Ac-YVAD.AMC (▪–▪)  hydrolysis activities were determined as described in Materials  and Methods and are shown in A. The extent of apoptosis at each  time point, as assessed by flow cytometry, is indicated in brackets.  Lysates from both untreated (Con) and THP.1 cells exposed to  an apoptotic stimulus (CHX + TLCK) were analyzed by Western  blot analysis using antibodies to CPP32 as described in Materials and Methods. (B) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower  arrowhead. The time course of cleavage of CPP32 (B) paralleled  the hydrolysis activity of the lysates towards Z-DEVD.AFC.
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Figure 5: Z.DEVD-AFC cleavage activity in lysates derived from THP.1 cells committed to apoptosis precedes the morphological characteristics of cell death. THP.1 cells were incubated for up to 4 h, either alone (Con) (○–○) or in the presence of cycloheximide (CHX) and TLCK (•–•; ▪–▪), and lysates were prepared at the indicated time points as described in Materials and Methods. Z-DEVD.AFC (○–○; •–•) and Ac-YVAD.AMC (▪–▪) hydrolysis activities were determined as described in Materials and Methods and are shown in A. The extent of apoptosis at each time point, as assessed by flow cytometry, is indicated in brackets. Lysates from both untreated (Con) and THP.1 cells exposed to an apoptotic stimulus (CHX + TLCK) were analyzed by Western blot analysis using antibodies to CPP32 as described in Materials and Methods. (B) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead. The time course of cleavage of CPP32 (B) paralleled the hydrolysis activity of the lysates towards Z-DEVD.AFC.
Mentions: The processing/cleavage of CPP32, Ich-1, Mch2α, PARP, U1-70K, and lamins A/B strongly suggested that activation of an ICE-like proteolytic activity was an important effector of the cell death program. This was further investigated using lysates from unsorted control and apoptotic cells to assay for proteolytic activity with Z-DEVD.AFC. This model substrate was chosen because the DEVD tetrapeptide sequence is identical to the cleavage site of PARP, i.e., DEVD216-G217. In addition, CPP32 (Nicholson et al., 1995), Mch2α, and Mch3α (Fernandes-Alnemri et al., 1995a,b) cleave the similar substrate analogue, Ac-DEVD.AMC. The Z-DEVD.AFC cleaving activity of lysates obtained from control cells was very low and did not increase after incubation of cells for 4 h (Fig. 5 A). In contrast, increased proteolytic activity was observed in lysates prepared from cells exposed to an apoptotic stimulus (Fig. 5 A). Within 1 h of exposure to the apoptotic stimulus, a small increase in Z-DEVD.AFC cleavage activity was detected in lysates (Fig. 5 A), which preceded the onset of apoptosis in intact cells, as measured by flow cytometry (Figs. 1 A and 5 A). The maximal increase in proteolytic activity occurred at 2 h and was ∼15-fold greater than the activity in control lysates (Fig. 5 A).

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