Limits...
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.

Show MeSH

Related in: MedlinePlus

Z-VAD.FMK inhibits the cleavage of CPP32 and Ich-1.  THP.1 cells were incubated for 4 h, either alone (lane 1) or with  etoposide (25 μM) in the presence (lane 3) or absence (lane 2) of  Z-VAD.FMK (50 μM) as described in Materials and Methods.  CPP32 and Ich-1 were detected by Western blot analysis. (Upper  panel) pro-CPP32 is indicated by the upper arrowhead, and the  17-kD cleavage product is indicated by the lower arrowhead;  (lower panel) 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower  arrowhead. On induction of apoptosis, intact CPP32 (upper  panel) and Ich-1 (lower panel) in control cells (lane 1) were  cleaved (lane 2), and these were inhibited by Z-VAD.FMK (lane 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139780&req=5

Figure 3: Z-VAD.FMK inhibits the cleavage of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with etoposide (25 μM) in the presence (lane 3) or absence (lane 2) of Z-VAD.FMK (50 μM) as described in Materials and Methods. CPP32 and Ich-1 were detected by Western blot analysis. (Upper panel) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead; (lower panel) 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead. On induction of apoptosis, intact CPP32 (upper panel) and Ich-1 (lower panel) in control cells (lane 1) were cleaved (lane 2), and these were inhibited by Z-VAD.FMK (lane 3).

Mentions: Incubation of THP.1 cells for 4 h with etoposide (25 μM) resulted in the induction of apoptosis (33.5 ± 2.5%, mean ± SEM, n = 3). Etoposide also induced the processing of the proforms of both CPP32 and Ich-1. This accompanied the appearance of the catalytically active p17 subunit of CPP32 and the p12 subunit of Ich-1, both of which were apparent when compared with control cells (Fig. 3, compare lanes 1 and 2). Pretreatment for 1 h with Z-VAD.FMK (50 μM) inhibited not only apoptosis (1.7 ± 0.5%) but also the loss of both pro–Ich-1 and pro-CPP32 and the formation of the p17 subunit of CPP32 and the p12 subunit of Ich-1 (Fig. 3, lanes 2 and 3). Pretreatment of THP.1 cells for 1 h with Z-VAD.FMK also completely prevented etoposide-induced cleavage of PARP, U1-70K, and lamins A/B (data not shown). These results demonstrated that the proforms of both CPP32 and Ich-1 were processed in THP.1 cells undergoing apoptosis to yield their catalytically active subunits, and that inhibition of their processing by Z-VAD.FMK corresponded with an inhibition 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)

Z-VAD.FMK inhibits the cleavage of CPP32 and Ich-1.  THP.1 cells were incubated for 4 h, either alone (lane 1) or with  etoposide (25 μM) in the presence (lane 3) or absence (lane 2) of  Z-VAD.FMK (50 μM) as described in Materials and Methods.  CPP32 and Ich-1 were detected by Western blot analysis. (Upper  panel) pro-CPP32 is indicated by the upper arrowhead, and the  17-kD cleavage product is indicated by the lower arrowhead;  (lower panel) 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower  arrowhead. On induction of apoptosis, intact CPP32 (upper  panel) and Ich-1 (lower panel) in control cells (lane 1) were  cleaved (lane 2), and these were inhibited by Z-VAD.FMK (lane 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139780&req=5

Figure 3: Z-VAD.FMK inhibits the cleavage of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with etoposide (25 μM) in the presence (lane 3) or absence (lane 2) of Z-VAD.FMK (50 μM) as described in Materials and Methods. CPP32 and Ich-1 were detected by Western blot analysis. (Upper panel) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead; (lower panel) 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead. On induction of apoptosis, intact CPP32 (upper panel) and Ich-1 (lower panel) in control cells (lane 1) were cleaved (lane 2), and these were inhibited by Z-VAD.FMK (lane 3).
Mentions: Incubation of THP.1 cells for 4 h with etoposide (25 μM) resulted in the induction of apoptosis (33.5 ± 2.5%, mean ± SEM, n = 3). Etoposide also induced the processing of the proforms of both CPP32 and Ich-1. This accompanied the appearance of the catalytically active p17 subunit of CPP32 and the p12 subunit of Ich-1, both of which were apparent when compared with control cells (Fig. 3, compare lanes 1 and 2). Pretreatment for 1 h with Z-VAD.FMK (50 μM) inhibited not only apoptosis (1.7 ± 0.5%) but also the loss of both pro–Ich-1 and pro-CPP32 and the formation of the p17 subunit of CPP32 and the p12 subunit of Ich-1 (Fig. 3, lanes 2 and 3). Pretreatment of THP.1 cells for 1 h with Z-VAD.FMK also completely prevented etoposide-induced cleavage of PARP, U1-70K, and lamins A/B (data not shown). These results demonstrated that the proforms of both CPP32 and Ich-1 were processed in THP.1 cells undergoing apoptosis to yield their catalytically active subunits, and that inhibition of their processing by Z-VAD.FMK corresponded with an inhibition 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.

Show MeSH
Related in: MedlinePlus