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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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CPP32 and Ich-1 are  extensively cleaved in apoptotic  but not morphologically normal  THP.1 cells, and their processing  is concomitant with the cleavage  of PARP, U1-70K, and lamins A/ 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  described previously (Zhu et al.,  1995). Cells with low blue fluorescence were morphologically  normal and those with high blue  fluorescence exhibited distinctive apoptotic morphology when  examined by fluorescence microscopy. Cells with either normal  (lane 1) or apoptotic (lane 2)  morphology were analyzed by  Western blot analysis as described in Materials and Methods. (A) Cells were analyzed using antibodies to CPP32 (upper  panel) and Ich-1 (lower panel).  (B) Cells were analyzed using antibodies to PARP, U1-70K, and  lamins A/B (upper, middle and  lower panels, respectively). The  proforms of CPP32 and Ich-1 are  indicated by the upper arrowheads (A), and intact PARP, U170K, and lamins A/B are indicated by the upper arrowheads  (B). The lower arrowheads represent either processed enzymes or cleaved proteolytic fragments. Cells displaying normal morphology  contain primarily the intact forms of all the proteins analyzed (lane 1), whereas, in apoptotic cells, processing of more than one ICE-like  protease is detected and associated with cleavage of PARP, U1-70K, and lamins A/B.
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Figure 2: CPP32 and Ich-1 are extensively cleaved in apoptotic but not morphologically normal THP.1 cells, and their processing is concomitant with the cleavage of PARP, U1-70K, and lamins A/ 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 described previously (Zhu et al., 1995). Cells with low blue fluorescence were morphologically normal and those with high blue fluorescence exhibited distinctive apoptotic morphology when examined by fluorescence microscopy. Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis as described in Materials and Methods. (A) Cells were analyzed using antibodies to CPP32 (upper panel) and Ich-1 (lower panel). (B) Cells were analyzed using antibodies to PARP, U1-70K, and lamins A/B (upper, middle and lower panels, respectively). The proforms of CPP32 and Ich-1 are indicated by the upper arrowheads (A), and intact PARP, U170K, and lamins A/B are indicated by the upper arrowheads (B). The lower arrowheads represent either processed enzymes or cleaved proteolytic fragments. Cells displaying normal morphology contain primarily the intact forms of all the proteins analyzed (lane 1), whereas, in apoptotic cells, processing of more than one ICE-like protease is detected and associated with cleavage of PARP, U1-70K, and lamins A/B.

Mentions: As apoptosis is a stochastic process, cells at all stages of the apoptotic process, including normal, condemned, and apoptotic, are present after exposure to an apoptotic stimulus (Earnshaw, 1995). Thus, the data describing the status of the ICE-like proteases in Fig. 1 and in all similar studies in the literature refer to results obtained with a mixed population of cells. To overcome this problem, we have used a flow cytometric method to sort pure populations of normal and apoptotic cells for further analysis (Zhu et al., 1995). 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. Dramatic differences were observed in the level of the proforms of both CPP32 and Ich-1 between these normal and apoptotic cells. Cells displaying normal morphology contained only the proform of CPP32 and predominantly the proform of Ich-1, whereas apoptotic cells contained almost no pro-CPP32 or pro–Ich-1 (Fig. 2 A, compare lanes 1 and 2). In the case of CPP32, the catalytically active subunit p17 was detected in apoptotic cells but not in normal cells (Fig. 2 A, compare lanes 1 and 2). For Ich-1, significant levels of the p12 subunit were detected in apoptotic cells with only very low levels detected in normal cells (Fig. 2 A). As low levels of the p12 subunit were observed in morphologically normal cells obtained after exposure to an apoptotic stimulus, we examined similar morphologically normal cells, which had not been exposed to an apoptotic stimulus. No p12 subunit of Ich-1 was seen in these sorted cells (data not shown). No cleavage of CPP32 was observed until cells displaying normal morphology (low Hoechst fluorescence) acquired apoptotic morphology (high blue fluorescence). In contrast, a very small amount of cleavage of Ich-1 was evident in morphologically normal cells exposed to an apoptotic stimulus, with extensive cleavage only observed when these cells acquired apoptotic morphology. Thus, extensive cleavage/activation of at least two ICE homologues 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)

CPP32 and Ich-1 are  extensively cleaved in apoptotic  but not morphologically normal  THP.1 cells, and their processing  is concomitant with the cleavage  of PARP, U1-70K, and lamins A/ 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  described previously (Zhu et al.,  1995). Cells with low blue fluorescence were morphologically  normal and those with high blue  fluorescence exhibited distinctive apoptotic morphology when  examined by fluorescence microscopy. Cells with either normal  (lane 1) or apoptotic (lane 2)  morphology were analyzed by  Western blot analysis as described in Materials and Methods. (A) Cells were analyzed using antibodies to CPP32 (upper  panel) and Ich-1 (lower panel).  (B) Cells were analyzed using antibodies to PARP, U1-70K, and  lamins A/B (upper, middle and  lower panels, respectively). The  proforms of CPP32 and Ich-1 are  indicated by the upper arrowheads (A), and intact PARP, U170K, and lamins A/B are indicated by the upper arrowheads  (B). The lower arrowheads represent either processed enzymes or cleaved proteolytic fragments. Cells displaying normal morphology  contain primarily the intact forms of all the proteins analyzed (lane 1), whereas, in apoptotic cells, processing of more than one ICE-like  protease is detected and associated with cleavage of PARP, U1-70K, and lamins A/B.
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Figure 2: CPP32 and Ich-1 are extensively cleaved in apoptotic but not morphologically normal THP.1 cells, and their processing is concomitant with the cleavage of PARP, U1-70K, and lamins A/ 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 described previously (Zhu et al., 1995). Cells with low blue fluorescence were morphologically normal and those with high blue fluorescence exhibited distinctive apoptotic morphology when examined by fluorescence microscopy. Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis as described in Materials and Methods. (A) Cells were analyzed using antibodies to CPP32 (upper panel) and Ich-1 (lower panel). (B) Cells were analyzed using antibodies to PARP, U1-70K, and lamins A/B (upper, middle and lower panels, respectively). The proforms of CPP32 and Ich-1 are indicated by the upper arrowheads (A), and intact PARP, U170K, and lamins A/B are indicated by the upper arrowheads (B). The lower arrowheads represent either processed enzymes or cleaved proteolytic fragments. Cells displaying normal morphology contain primarily the intact forms of all the proteins analyzed (lane 1), whereas, in apoptotic cells, processing of more than one ICE-like protease is detected and associated with cleavage of PARP, U1-70K, and lamins A/B.
Mentions: As apoptosis is a stochastic process, cells at all stages of the apoptotic process, including normal, condemned, and apoptotic, are present after exposure to an apoptotic stimulus (Earnshaw, 1995). Thus, the data describing the status of the ICE-like proteases in Fig. 1 and in all similar studies in the literature refer to results obtained with a mixed population of cells. To overcome this problem, we have used a flow cytometric method to sort pure populations of normal and apoptotic cells for further analysis (Zhu et al., 1995). 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. Dramatic differences were observed in the level of the proforms of both CPP32 and Ich-1 between these normal and apoptotic cells. Cells displaying normal morphology contained only the proform of CPP32 and predominantly the proform of Ich-1, whereas apoptotic cells contained almost no pro-CPP32 or pro–Ich-1 (Fig. 2 A, compare lanes 1 and 2). In the case of CPP32, the catalytically active subunit p17 was detected in apoptotic cells but not in normal cells (Fig. 2 A, compare lanes 1 and 2). For Ich-1, significant levels of the p12 subunit were detected in apoptotic cells with only very low levels detected in normal cells (Fig. 2 A). As low levels of the p12 subunit were observed in morphologically normal cells obtained after exposure to an apoptotic stimulus, we examined similar morphologically normal cells, which had not been exposed to an apoptotic stimulus. No p12 subunit of Ich-1 was seen in these sorted cells (data not shown). No cleavage of CPP32 was observed until cells displaying normal morphology (low Hoechst fluorescence) acquired apoptotic morphology (high blue fluorescence). In contrast, a very small amount of cleavage of Ich-1 was evident in morphologically normal cells exposed to an apoptotic stimulus, with extensive cleavage only observed when these cells acquired apoptotic morphology. Thus, extensive cleavage/activation of at least two ICE homologues 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