* Medical Research Council Toxicology Unit, Centre for Mechanisms of Human Toxicity, University of Leicester, Leicester
LE1 9HN, United Kingdom; and Centre for Apoptosis Research and Kimmel Cancer Institute, Jefferson Medical College,
Philadelphia, Pennsylvania 19107
Identification of the processing/activation of
multiple interleukin-1 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 Mch3
to their catalytically active subunits,
and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3
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 Mch3
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 (U170K), and lamins A/B. In contrast, no cleavage of
CPP32, Mch3
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, Mch3
,
Mch2
, 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,
Mch3
, and Mch2
. Together these observations demonstrate that processing/activation of Ich-1, CPP32,
Mch3
, and Mch2
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.
Apoptosis is a form of cell death that is essential for
the control of cell populations during normal development and in many diseases (Arends and
Wyllie, 1991 In the nematode, Caenorhabditis elegans genetically determined cell death has an essential requirement for the
gene ced-3 (Ellis et al., 1991 While initial studies highlighted a major role for ICE in
apoptosis, recent investigations demonstrated that mice
deficient in ICE fail to exhibit a prominent cell death-
defective phenotype, except in Fas-induced apoptosis of thymocytes (Li et al., 1995 ICE-like proteases are synthesized as inactive precursors requiring cleavage at specific Asp residues to yield
two subunits of molecular mass ~20 and 10 kD, which together form the active enzyme (Thornberry et al., 1992 It remains to be determined whether activation of more
than one ICE-like protease is required for the execution of
apoptosis (Takahashi and Earnshaw, 1996 Materials
Media and serum were purchased from Gibco (Paisley, UK). Pronase and
N- Cell Culture
The human monocytic tumor cell line, THP.1, was obtained from ECACC
(Wiltshire, UK) and maintained in RPMI 1640 supplemented with 10%
heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in
an atmosphere of 5% CO2 in air at 37°C. The cells were maintained in logarithmic growth phase by routine passage every 2-3 d. To induce apoptosis, 1 × 10 6 cells per ml were incubated either alone or in the presence of cycloheximide (25 µM) and TLCK (100 µM) or etoposide (25 µM) as previously described (Zhu et al., 1995 Preparation of Cell and Bacterial Lysates
For preparation of lysates, THP.1 cells were incubated as required and
then placed on ice, washed twice with ice-cold PBS, and resuspended in
Pipes buffer (50 mM Pipes/KOH (pH 6.5), 2 mM EDTA, 0.1% (wt/vol)
CHAPS, 5 mM DTT, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml
aprotinin, and 2 mM PMSF) at a concentration of 6 × 10 6 cells per 10 µl.
The cells were frozen and thawed three times in liquid nitrogen, and then
centrifuged at 20,000 g for 30 min at 4°C. The supernatant fraction was
then centrifuged for an additional 45 min at 100,000 g. The protein concentration in the supernatant fractions (the lysate) was determined by
the Bradford assay (Bio Rad Laboratories, Hercules, CA). Recombinant
CPP32 and Mch3 Separation of Normal and Apoptotic Cells by
Flow Cytometry
Apoptosis was assessed by flow cytometry using Hoechst 33342 as previously described (Sun et al., 1992 Western Blot Analysis
Samples of 0.5 × 106 cells were prepared as described (Harlow and Lane,
1988 Fluorimetric Measurement of Proteolytic Cleavage
The proteolytic cleavage activities of THP.1 lysates and recombinant
CPP32 and Mch3 Generation of a Polyclonal Antibody to Mch3 A polyclonal antibody to Mch3 Processing of CPP32 and Ich-1 Accompanies the
Induction of Apoptosis in Intact THP.1 Cells
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
Extensive Processing of CPP32 and Ich-1 in
Apoptotic THP.1 Cells But Not in Cells That Exhibit
Normal Morphology
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
Cleavage of PARP, U1-70K, and Lamins A/B
Accompanied the Processing of CPP32 and Ich-1 in
Apoptotic Cells
To obtain further evidence for the activation of more than
one ICE-like protease during apoptosis, we used antibodies to PARP, U1-70K, and lamins A/B to determine which
putative ICE-like protease substrates were cleaved in apoptotic cells. Pure populations of normal and apoptotic cells
were sorted by flow cytometry as described in the previous
section. In cells displaying normal morphology, only the intact form of all three proteins was detected, namely 116-kD
PARP protein, 64-kD U1-70K protein, and 66-kD lamin A/B protein (Fig. 2 B, lane 1). In marked contrast, in a pure
population of apoptotic cells, all three proteins were processed, resulting in their disappearance together with the
appearance of, in the case of PARP and lamin A/B, protein fragments of 85 and 46 kD, respectively (Fig. 2 B,
lane 2). Therefore, several target proteins, which previously have been reported to be substrates for ICE-like proteases, are cleaved in the execution phase of apoptosis.
Cleavage of PARP and U1-70K suggested activation of
CPP32 and/or Mch3 Z-VAD.FMK Inhibits Both Apoptosis and the
Processing of CPP32 and Ich-1
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.
YVAD.CMK Inhibits Lamin Cleavage But
Not Apoptosis
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
Mch2 The ability of YVAD.CMK to inhibit lamin cleavage, but
not other features of apoptosis, provided indirect evidence
for activation of the lamin protease, Mch2 Induction of Apoptosis Activates Z-DEVD.AFC
Cleavage in Cell Lysates
The processing/cleavage of CPP32, Ich-1, Mch2
We wished to investigate whether these changes in proteolytic activity correlated with cleavage of CPP32. Lysates obtained from control cells incubated for 4 h contained only the proform of CPP32 (Fig. 5 B, lane 1). In
contrast, lysates from cells exposed to an apoptotic stimulus displayed a time-dependent loss of the proform of
CPP32 (Fig. 5 B, lanes 2-4). This was accompanied by the
appearance of the p17 subunit of CPP32, which was first
observed at 1 h, was maximal at 2 h, but decreased at 4 h
(Fig. 5 B). In the same lysates, cleavage or loss of the proform of Ich-1 was almost maximal at 1 h, the first time
point examined (data not shown).
In the present study lysates obtained from THP.1 cells
that had been pretreated with Z-VAD.FMK before induction of apoptosis exhibited neither Z-DEVD.AFC proteolytic activity nor cleavage of CPP32 or Ich-1 (data not
shown). Thus, Z-VAD.FMK inhibits the generation of a
CPP32-like protease activity in addition to blocking the
cleavage of both Ich-1 and CPP32.
Induction of Apoptosis in THP.1 Cells Results in
Cleavage of Z-DEVD.AFC But Not of Ac-YVAD.AMC
To further characterize the proteolytic activity in THP.1
cells lysates, we investigated its susceptibility to specific
inhibitors. Ac-YVAD.CHO is a specific inhibitor of ICE
(Thornberry et al., 1992 Table I.
Kinetic Properties of Lysates Prepared from THP.1
Cells Exposed to an Apoptotic Stimulus
; Raff, 1992
). It is recognized by distinct morphological changes, including cell shrinkage, nuclear condensation, and fragmentation. Apoptotic cell death has
been divided into two distinct phases: an initial condemned
phase where cells receive a signal that results in commitment to cell death without any morphological changes, followed by an execution phase when all of the characteristic
morphological and biochemical features of apoptosis occur (Earnshaw, 1995
).
), which encodes a protein with
sequence homology to the mammalian cysteine protease,
interleukin-1
converting enzyme (ICE)1 (Yuan et al.,
1993
). ICE is required for the proteolytic processing of
pro-interleukin-1
to the active cytokine (Thornberry et al., 1992
). Overexpresssion of ICE-like proteases induces cell
death by apoptosis (for review see Kumar, 1995
), suggesting that ICE, or a related protease, is an essential component of the mammalian cell death pathway. However, such
studies have to be interpreted with some caution as overexpression of a particular protease may result in the cleavage of low affinity protein substrates leading to cytotoxicity. More direct evidence for the involvement of ICE or
ICE-like proteases in apoptosis has come from inhibitor
studies with CrmA, a viral serpin inhibitor of ICE (Ray et al.,
1992
), and the baculovirus antiapoptotic protein p35 (Bump
et al., 1995
).
; Kuida et al., 1995
). This suggested
that other ICE homologues may be required for apoptosis,
and recent work has identified a family of such proteases
including CPP32/apopain/Yama (Fernandes-Alnemri et
al., 1994
; Nicholson et al., 1995
; Tewari et al., 1995
), Ich-1 (Wang et al., 1994
) and its mouse homologue Nedd-2 (Kumar et al., 1994
), Mch2 (Fernandes-Alnemri et al., 1995a
),
Mch3/ ICE-LAP3 (Duan et al., 1996
; Fernandes-Alnemri
et al., 1995b
), Mch4 (Fernandes-Alnemri et al., 1996
),
MACH/FLICE/ Mch5 (Boldin et al., 1996
; FernandesAlnemri et al., 1996; Muzio et al., 1996
), Mch6 (Srinivasula et al., 1996
), Tx/Ich-2/ICErel-II (Faucheu et al., 1995
;
Kamens et al., 1995
; Munday et al., 1995
), and ICErel-III/
Ty (Munday et al., 1995
; Faucheu et al., 1996).
;
Thornberry and Molineaux, 1995
). Several studies have
suggested that ICE-like proteases may autoprocess or,
alternatively, some ICE-like proteases may activate other family members (for review see Takahashi and Earnshaw,
1996
). During apoptosis, ICE-like proteases cleave several
intracellular proteins including poly (ADP-ribose) polymerase (PARP), lamins, the 70-kD protein component of U1
small nuclear ribonucleoprotein (U1-70K), DNA-dependent protein kinase, and sterol regulatory element binding
proteins (Casciola-Rosen et al., 1994
, 1996; Kaufmann et al.,
1993
; Song et al., 1996
; Wang et al., 1995
). Although the
precise substrate specificity of different ICE homologues is not known, CPP32, Mch2
, and Mch3
cleave PARP
(Nicholson et al., 1995
; Fernandes-Alnemri et al., 1995a
,b;
Lazebnik et al., 1995
), while Mch2
but not CPP32 can
cleave lamins (Orth et al., 1996
; Takahashi et al., 1996
), and
CPP32 can cleave U1-70K (Casciola-Rosen et al., 1996
).
). In the present
study we have used peptide inhibitors to distinguish the potential roles of different ICE-like proteases in apoptosis.
Acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD.CHO) and
acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD.CHO) are
specific inhibitors of ICE (Thornberry et al., 1992
) and
CPP32/Mch3
, respectively (Fernandes-Alnemri et al.,
1995b
; Nicholson et al., 1995
). Although acetyl-Tyr-ValAla-Asp chloromethyl ketone (YVAD.CMK) was originally described as a specific inhibitor of ICE, it also inhibits other ICE-like proteases, including the proteolytic
activity described as prICE (Lazebnik et al., 1994
). Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone
(Z-VAD.FMK), a cell membrane-permeable inhibitor of
ICE-like proteases, inhibits apoptosis in diverse systems
including human monocytic tumor THP.1 cells (Chow et al., 1995
; Fearnhead et al., 1995
; Zhu et al., 1995
). Z-VAD. FMK
inhibits apoptosis at an early stage, as evidenced by inhibition of DNA fragmention, PARP proteolysis (Zhu et al.,
1995
), and processing of CPP32 (Jacobson et al., 1996
; Slee
et al., 1996
). Using these inhibitors, we now demonstrate
the cleavage/activation of at least four ICE-like proteases,
Ich-1, CPP32, Mch3
, and Mch2
, in THP.1 cells during
the execution phase of apoptosis.
Materials and Methods
-p-tosyl-L-lysine chloromethyl ketone (TLCK) were from BoehringerMannheim UK (Lewes, UK). Z-VAD.FMK and benzyloxycarbonylAsp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (Z-DEVD.AFC)
were from Enzyme Systems, Inc. (Dublin, CA). Ac-YVAD.CHO,
Ac-DEVD.CHO, and YVAD.CMK were from Bachem (Bubendorf, Switzerland). Acetyl-Tyr-Val-Ala-Asp-7-amino-4-methyl coumarin (AcYVAD.AMC) and all other chemicals were from Sigma Chemical Co.
(Poole, UK).
). Both these stimuli induce extensive
apoptosis in THP.1 cells. To assess the effect of the ICE-like protease inhibitors, THP.1 cells were pretreated for 1 h with Z-VAD.FMK (50 µM)
or YVAD.CMK (5-25 µM) before exposure to the apoptotic stimulus.
The peptide inhibitors were prepared as stock solutions in DMSO, and aliquots were added to the control cultures.
were expressed in bacteria and bacterial extracts prepared as previously described (Fernandes-Alnemri et al., 1995a
,b).
; Zhu et al., 1995
). The basis of this
method is the increased permeability to Hoechst 33342 of the apoptotic
cells compared with normal cells (Ormerod et al., 1993
). We have previously shown that the high blue fluorescent cells (indicative of increased
Hoechst 33342 staining) are apoptotic based on a number of criteria including ultrastructure and analysis of DNA by conventional agarose gel
electrophoresis (Sun et al., 1992
; Zhu et al., 1995
). In the present study we
used fluorescence-activated cell sorting to separate pure populations of
normal and apoptotic cells. When examined by fluorescence microscopy,
cells with low blue fluorescence exhibited normal morphology, and those
with high blue fluorescence exhibited distinctive apoptotic morphology.
). Proteins were resolved on 13% (CPP32, Mch3
, Mch2
, and Ich-1)
or 7% (PARP, U1-70K, and lamins A/B) SDS polyacrylamide gels and
blotted onto nitrocellulose (Hybond-C extra; Amersham, Little Chalfont,
UK). Membranes were blocked before detection with rabbit polyclonal
antibodies directed to the p17 subunit of CPP32 (Nicholson et al., 1995
),
or to the p12 subunit of Mch2
(both kindly provided by D. Nicholson,
Merck Frosst, Quebec, Canada); a rabbit polyclonal antibody to the carboxy terminus of Ich-1L (Santa Cruz Biotechnology, Santa Cruz, CA); or a
rabbit polyclonal antibody directed to the p19 subunit of Mch3
(see below). The CPP32 antibody detects both pro-CPP32 and the p17 subunit;
the antibody to Ich-1 detects both pro-Ich-1 and the p12 subunit; and the
antibody to Mch3
detects both pro-Mch3
and the p19 subunit, while the
antibody to Mch2
detects only pro-Mch2
. The rabbit polyclonal antibodies to PARP (318) and U1-70K were kind gifts from Dr. G. Poirier
(Laval University, Quebec, Canada) and Dr. K. Luhrmann (University of
Marburg, Germany), respectively. The PARP antibody recognizes both
intact PARP (116 kD) and a cleavage product of 85 kD, whereas the U170K antibody detects only intact U1-70K, which, in THP.1 cells, was calculated to have a molecular mass of ~64 kD. A mouse mAb to lamins A/B,
which detects both intact (66 kD) and cleaved (46 kD) lamins, was kindly
provided by Dr. E. Nigg (University of Geneva, Switzerland). Detection was achieved using the appropriate secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG) conjugated to HRP and by enhanced chemiluminescence (Amersham).
were measured using a continuous fluorimetric assay
modified from the method of Thornberry (1994)
. Liberation of 7-amino-4trifluoromethylcoumarin from Z-DEVD.AFC as a measure of CPP32like activity was assayed at excitation and emission wavelengths of 400 and 505 nm, respectively. ICE activity was measured by the liberation of
7-amino-4-methylcoumarin from Ac-YVAD.AMC at excitation and emission wave lengths of 380 and 460 nm, respectively. Lysates were assayed at
37°C in a modified thermostatted cuvette holder in 1.25 ml of 100 mM
Hepes, 10% sucrose, 0.1% CHAPS, 10 mM DTT, pH 7.5, in an LS50B luminescence fluorimeter (Perkin-Elmer Corp., Norwalk, CT). Routinely,
assays contained 100 µg of lysate protein and 20 µM substrate. Calibration was carried out with standard solutions of 7-amino-4-trifluoromethylcoumarin and 7-amino-4-methylcoumarin. Lysates, from THP.1 cells exposed for 1 h to an apoptotic stimulus, were preincubated for 15 min with
various concentrations of inhibitors to ensure equilibrium, and the reaction, initiated by addition of Z-DEVD.AFC, was followed for 5 min. Substrates and inhibitors were added in DMSO, whose final concentration
never exceeded 1% and had no effect on enzymic activity.
was raised against the p17 subunit of recombinant Mch3
, obtained using a glutathione-S-transferase-Mch3
cDNA
(Fernandes-Alnemri et al., 1995). Briefly, the p17 fragment of Mch3
(amino
acids 54-198) was subcloned in-frame into the BamH1 site of the bacterial
expression vector pGEX2T (Pharmacia Biotech, Uppsala, Sweden). Bacterial extracts were prepared, and glutathione-S-transferase-p17 Mch3
protein was purified by glutathione-agarose beads (Sigma Chemical Co.)
before immunization. The rabbit polyclonal antibody obtained was characterized by ELISA and Western blot analysis, which verified that the antibody obtained recognized both intact Mch3
(~36 kD) and the p19 subunit but did not recognize CPP32 (data not shown).
Results
). 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).
Fig. 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.
[View Larger Version of this Image (25K GIF file)]
). 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.
Fig. 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.
[View Larger Versions of these Images (17 + 18K GIF file)]
(Casciola-Rosen et al., 1996
; Fernandes-Alnemri et al., 1995b
; Nicholson et al., 1995
).
However, as Mch2
is the only ICE-like protease known
to cleave lamins (Orth et al., 1996
; Takahashi et al., 1996
),
the cleavage of lamins A/B in apoptotic cells supports the
activation of Mch2
in THP.1 cell apoptosis.
Fig. 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).
[View Larger Version of this Image (33K GIF file)]
). 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).
Fig. 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 Mch2.
[View Larger Versions of these Images (23 + 27K GIF file)]
Is Cleaved during the Execution
Phase of Apoptosis and This Cleavage Is Inhibited
by Z-VAD.FMK
(Orth et al.,
1996
; Takahashi et al., 1996
). To obtain direct evidence for
the involvement of Mch2
in THP.1 cell apoptosis, we
used an antibody that recognizes pro-Mch2
. Immunoblots showed the presence of the 34-kD pro-Mch2
in control cells incubated for 4 h (Fig. 4 C, lane 1). Induction of
apoptosis by cycloheximide in the presence of TLCK resulted in extensive loss of pro-Mch2
(Fig. 4 C, lane 2),
which was totally prevented by the ICE-like protease inhibitor, Z-VAD.FMK (Fig. 4 C, lane 3). These data demonstrate the activation of Mch2
during the execution phase
of apoptosis in THP.1 cells. Concentrations of YVAD.CMK
(25 µM), which did not inhibit apoptosis, did not significantly inhibit cleavage of pro-Mch2
(Fig. 4 C, lane 4). As
this concentration of YVAD.CMK inhibited lamin cleavage (Fig. 4 B), our data suggested that YVAD.CMK preferentially inhibited Mch2
activity rather than the processing of Mch2
.
, 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
), Mch2
, and Mch3
(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).
Fig. 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.
[View Larger Version of this Image (14K GIF file)]
), and Ac-DEVD.CHO is a relatively specific inhibitor of CPP32 (Nicholson et al., 1995
)
although it also inhibits Mch3
(Fernandes-Alnemri et al.,
1995b
) and Mch4 (Fernandes-Alnemri et al., 1996
). Hydrolysis of Z-DEVD.AFC was inhibited in a time-dependent manner by Ac-DEVD.CHO, Z-VAD.FMK, and AcYVAD.CHO (data not shown). When IC50 values were determined from the inhibition curves (Table I), AcDEVD.CHO was the most potent inhibitor with an IC50 of
3 nM. Z-VAD.FMK was a more potent inhibitor than
Ac-YVAD.CHO, but it was not as effective as AcDEVD.CHO (Table I). Ac-YVAD.CHO was 10,000-fold
less potent than Ac-DEVD.CHO. These differing potencies suggested that the ICE-like protease activity in the lysate was due to a CPP32-like activity but not to ICE. Further evidence was provided by our finding that these
lysates did not exhibit an increased ability to hydrolyze
Ac-YVAD.AMC, a model substrate for ICE activity (Fig. 5 A). These data were consistent with the Z-DEVD.AFC
hydrolysis activity of lysates, from cells exposed to an apoptotic stimulus, being due to a CPP32-like activity but not to
ICE.
Kinetic Characterization of Z-DEVD.AFC Cleavage
To further characterize this CPP32-like proteolytic activity, we investigated its kinetic characteristics. Lysates, from
cells exposed for 1 h to cycloheximide and TLCK, were incubated with Z-DEVD.AFC (10-200 µM) and proteolysis
was assessed. The lysates exhibited Michaelis-Menten kinetics with a Km of 55.4 ± 7.1 µM (mean ± SEM) and Vmax
= 411 ± 19.7 pmol/mg/min (mean ± SEM). This Km is higher
than that for pure or recombinant CPP32 (FernandesAlnemri et al., 1995b; Nicholson et al., 1995) and is very
similar to that reported for recombinant Mch3
(FernandesAlnemri et al., 1995b). To determine whether lysates, obtained from cells exposed to an apoptotic stimulus, contained a proteolytic activity characteristic of both CPP32
and Mch3
, the kinetic characteristics of these lysates were
compared with those of recombinant CPP32 and Mch3
. Bacterial extracts of CPP32 and Mch3
were incubated
with Z-DEVD.AFC (10-200 µM) and proteolysis was assessed. Both CPP32 and Mch3
exhibited MichaelisMenten kinetics with a Km of 21.9 ± 2.0 µM and 61.5 ± 8.8 µM, respectively. Thus, lysates, obtained from THP.1 cells
exposed to an apoptotic stimulus, exhibited kinetic characteristics suggestive of the activation of both CPP32 and Mch3
in the execution phase of apoptosis in THP.1 cells.
Processing of Mch3 during the Execution
Phase of Apoptosis Is Inhibited by ZVAD.FMK But
Not by YVAD.CMK
Characterization of the proteolytic activity present in lysates suggested the activation of another CPP32-like protease, in addition to CPP32 itself, in the execution phase of
apoptosis in THP.1 cells. Kinetic data suggested that this
protease was Mch3, and to verify we raised an antibody
to the p17 subunit of Mch3
. THP.1 cells were coincubated with cycloheximide (25 µM) and TLCK (100 µM)
for up to 4 h, and the cleavage of Mch3
was assessed (Fig.
6 A). In untreated cells, immunoblots showed the presence
of the 34-kD precursor form of Mch3
(Fig. 6 A, lane 1).
After induction of apoptosis, the p19 subunit of Mch3
was first detected by 2 h and remained evident until 4 h
(Fig. 6 A, lanes 2-5). In parallel, a time-dependent decrease in the 34-kD proform of Mch3
was observed, coincident with the induction of apoptosis as previously assessed by flow cytometry (Fig. 1 A). Similar results were
obtained when apoptosis was induced by etoposide (data
not shown). To determine whether processing of Mch3
correlated with apoptotic execution, pure populations of
normal and apoptotic cells were obtained and analyzed.
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.
Cells displaying normal morphology contained only the
proform of Mch3
(Fig. 6 B, lane 1), whereas apoptotic
cells contained the catalytically active subunit p19 with almost no intact Mch3
(Fig. 6 B, lane 2). Thus, no cleavage
of Mch3
was observed until cells displaying normal morphology acquired apoptotic morphology, suggesting that
activation of Mch3
had occurred during the execution
phase of apoptosis.
To investigate whether the ability of Z-VAD.FMK to
inhibit apoptosis (see above) corresponded with an inhibition of the processing of Mch3, THP.1 cells were pretreated for 1 h with ZVAD.FMK (50 µM), and then coincubated with cycloheximide (25 µM) and TLCK (100 µM)
for 4 h. Pretreatment with Z-VAD.FMK inhibited both
the processing of proMch3
and the formation of the catalytically active p19 subunit (Fig. 6 A, lane 6). Pretreatment
of THP.1 cells for 1 h with YVAD.CMK (25 µM) before
treatment with etoposide (25 µM) did not inhibit apoptosis (see above) or the processing of the proform of Mch3
(Fig. 6 C, compare lanes 2 and 3). These results demonstrated that processing of the proform of Mch3
to yield
the catalytically active subunit p19 occurred in THP.1 cells undergoing apoptosis, and that inhibition of this processing was concomitant with an inhibition of apoptosis.
Processing/Activation of At Least Four ICE-like Proteases Accompanies the Execution of Apoptosis
Apoptosis in THP.1 cells was accompanied by processing
of the proforms of three ICE homologues, Ich-1, CPP32,
and Mch3, together with the appearance of their catalytically active p12, p17, and p19 subunits, respectively (Figs.
1 and 6). While we have demonstrated that the loss of
CPP32 and Mch3
was due to processing to an active protease (Fig. 5), we cannot state unequivocally that Ich-1 was similarly activated, as an intracellular substrate for Ich-1
has yet to be identified. However, cleavage of Ich-1 to
yield its p12 subunit and the ability of Z-VAD.FMK to
prevent the loss of Ich-1 (Fig. 3) suggested that Ich-1 was
also cleaved by an ICE-like protease to an active enzyme.
This is the first demonstration of the processing of Ich-1 in
apoptotic cells; thus the identification/development of either natural or synthetic substrates is required to further elucidate the role(s) of Ich-1 in apoptosis. In this regard, it is significant that Z-DEVD.AFC is a very poor substrate
for recombinant Ich-1/Nedd2 (Kumar, S., personal communication).
The data derived from pure populations of morphologically normal and apoptotic cells, obtained by fluorescenceactivated cell sorting (Figs. 2 and 6), provided further
evidence for the involvement of at least four ICE-like proteases in apoptosis in THP.1 cells. Normal cells contained
only the proforms of CPP32 and Mch3 and predominantly pro-Ich-1 together with intact PARP, U1-70K, and lamins A/B. In marked contrast, almost all of the Ich-1,
CPP32, and Mch3
was processed in apoptotic cells and
the ICE-like protease substrates were extensively cleaved
(Figs. 2 and 6). Although it is probable that different ICElike proteases may have overlapping substrate specificities, recent studies with cloned recombinant ICE-like proteases have shown that certain ICE-like proteases cleave
specific substrates. Of relevance to the present work, two
studies have shown that proteolysis of lamins is mediated by Mch2
but not by CPP32 or Mch3
(Orth et al., 1996
;
Takahashi et al., 1996
). Cleavage of PARP is mediated
primarily by CPP32/Mch3
with a possible contribution
from Mch2
(Nicholson et al., 1995
; Tewari et al., 1995
;
Fernandes-Alnemri et al., 1995a
,b), while cleavage of U170K is mediated by CPP32 (Casciola-Rosen et al., 1996
).
Thus, the cleavage in the apoptotic cells of PARP and U170K and in particular lamins A/B (Fig. 2) suggested the activation of Mch2
in addition to CPP32/Mch3
. Further
indirect evidence for the activation of Mch2
was provided by the sensitivity of the lamin protease to YVAD.
CMK (Fig. 4) (Lazebnik et al., 1995
). Direct evidence for
the activation of Mch2
during the execution phase of
apoptosis was provided by the loss of pro-Mch2
and its prevention by Z-VAD.FMK (Fig. 4 C). These results provide strong evidence for the activation of Ich-1, CPP32,
Mch3
, and Mch2
in apoptosis in intact THP.1 cells. This
study demonstrates for the first time the activation of at
least four ICE-like proteases during the execution phase
of apoptosis.
Z-VAD.FMK Inhibits Apoptosis and the Processing of Multiple ICE-like Proteases
The ability of Z-VAD.FMK to inhibit apoptosis in different cell systems (Chow et al., 1995; Fearnhead et al., 1995
;
Zhu et al., 1995
; Cain et al., 1996
; Jacobson et al., 1996
;
Pronk et al., 1996
; Slee et al., 1996
) has shown that this
compound is a valuable tool in the study of apoptosis. Its
precise site(s) of action is still unknown, but the present
study shows that both apoptosis and the processing of CPP32,
Ich-1, Mch3
, and Mch2
were blocked by Z-VAD.FMK
(Figs. 3, 4, and 6). As the most likely target for Z-VAD.FMK
is an ICE-like protease, it is probable that this unidentified
protease is responsible for activation of Ich-1, CPP32,
Mch3
, and/or Mch2
. It is notable that YVAD.CMK, designed as a specific inhibitor of ICE, did not significantly
inhibit the processing of Ich-1, CPP32, Mch3
, or Mch2
(Figs. 4, A and C, and 6 C), suggesting that the protease
upstream of Ich-1, CPP32, Mch3
, and/or Mch2
is not
ICE. It appears that Mch4 and/or Mch5/MACH/FLICE might be the most upstream proteases responsible for the
activation of multiple CED-3/ICE homologues (Boldin et
al., 1996
; Fernandes-Alnemri et al., 1996
; Muzio et al.,
1996
). For example, recombinant Mch4 and Mch5 can process both pro-CPP32 and pro-Mch3 (Fernandes-Alnemri et al., 1996
; unpublished observations), but whether this
occurs in intact cells undergoing apoptosis is not known.
Activation of MACH/FLICE occurs as a consequence of
tumor necrosis factor- and Fas receptor-mediated cell
death (Boldin et al., 1996
; Muzio et al., 1996
). It remains
to be determined whether other forms of apoptosis, such
as drug-induced apoptosis, require activation of MACH/
FLICE/Mch5 or other cellular homologues such as Mch4.
Lysates from Apoptotic Cells Contain a CPP32/
Mch3-like Protease Activity
Lysates, from THP.1 cells exposed to an apoptotic stimulus, exhibited Z-DEVD.AFC cleavage activity, which was
potently inhibited by Ac-DEVD.CHO, an inhibitor of
CPP32 and Mch3 (Nicholson et al., 1995
; FernandesAlnemri et al., 1995b) (Table I). Z-DEVD.AFC hydrolysis
activity was due, in part, to cleavage of CPP32, as shown
by the appearance of its catalytically active subunit p17 (Fig. 5). Using Z-DEVD.AFC as a substrate, the lysates
had a Km of 55 µM, which is intermediate between that for
recombinant CPP32 (21.9 µM) and Mch3 (61.5 µM). The
Km values found in this study for recombinant CPP32 and
Mch3 using Z-DEVD.AFC as a substrate were similar to
those reported using Ac-DEVD.AMC (Nicholson et al.,
1995
; Fernandes-Alnemri et al., 1995b
). Thus, our data
suggested that, in addition to CPP32, activation of at least
one other CPP32-like protease, most likely Mch-3
, accompanied the execution of apoptosis in THP.1 cells and
was responsible for the Z-DEVD.AFC cleavage activity
found in the lysates. Activation of Mch3
in THP.1 cell lysates was demonstrated by Western blot analysis (data not
shown). Thus, our data demonstrating activation of both
CPP32 and Mch3
in THP.1 cell apoptosis is in agreement
with a recent study showing processing of these homologues in apoptosis in Jurkat T cells (Chinnaiyan et al.,
1996
).
ICE Is Not Activated in the Execution Phase of Apoptosis in THP.1 Cells
Our data from both cell lysates and intact cells did not support the involvement of ICE per se in the execution phase
of apoptosis in THP.1 cells. Lysates, from THP.1 cells exposed to an apoptotic stimulus, did not exhibit any significant increase in YVAD.AMC cleaving activity (Fig. 5 A).
Two additional pieces of evidence suggested that, under
our experimental conditions, ICE itself was not activated
in THP.1 cells. First, no cleavage of ICE into its catalytically active p20/p10 subunits was observed with an antibody specific to the p10 subunit of ICE (data not shown). Second, YVAD.CMK, designed as a specific inhibitor of
ICE, did not inhibit apoptosis in THP.1 cells. Our results,
which demonstrate that the execution of apoptosis resulted in a CPP32/Mch3-like protease activity but not
ICE per se, are in agreement with others (Nicholson et al.,
1995
; Jacobson et al., 1996
; Schlegel et al., 1996
; Slee et al.,
1996
; Nett-Fiordalisi et al., 1996). Our observations do not
exclude a role for ICE in certain forms of apoptosis (Li et al.,
1995
; Yuan et al., 1993
) such as Fas-mediated apoptosis when a transient increase in ICE activity preceded the appearance of a CPP32/Mch3
-like activity (Enari et al., 1996
).
We have demonstrated activation of four ICE-like proteases during the execution phase of apoptosis in THP.1
cells. The inhibition of lamin cleavage, but not apoptosis,
by YVAD.CMK (Fig. 4 B) suggested that Mch2-induced
lamin cleavage occurred late in the apoptotic process, in
agreement with other studies (Lazebnik et al., 1995
; Takahashi et al., 1996
). This is supported by the observation that recombinant CPP32 activates pro-Mch2
(Srinivasula
et al.,1996). Two pieces of data suggested that Ich-1 is processed early in the apoptotic process: the p12 subunit was
observed after 1 h exposure to an apoptotic stimulus (Fig.
1 B), and a small amount of the p12 subunit was also detected in the morphologically normal cells obtained by fluorescence-activated cell sorting (Fig. 2 A). As we cannot
exclude the possibility that the Ich-1 antibody may be
more sensitive than the other antibodies, we cannot say whether Ich-1 is activated before CPP32 and/or Mch3
. It
remains to be determined whether activation of Ich-1 may
lead either directly or indirectly to activation of these
other homologues.
In summary, we demonstrate that processing/activation
of Ich-1, CPP32, Mch3, and Mch2
accompanies the generation of the apoptotic phenotype in THP.1 cells. Furthermore, we show that Z-VAD.FMK blocks apoptosis,
most likely through inhibition of an unidentified key effector ICE-like protease, possibly MACH/FLICE/Mch5 or
Mch4, thereby preventing cleavage/processing of Ich-1,
CPP32, Mch3
, and Mch2
. This is the first demonstration
of the activation of at least four ICE-like proteases during
the execution phase of apoptosis in intact cells.
Received for publication 10 October 1996 and in revised form 4 February 1997.
Please address all correspondence to Gerald M. Cohen, MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom. Tel.: (44) 116-252-5589/5600. Fax: (44) 116-252-5616. e-mail: gmc2{at}leicester.ac.ukWe thank R. Snowden for help with the flow cytometry.
This work was supported in part by research grant AG 13847 from the National Institutes of Health (to E.S. Alnemri).
ICE, interleukin-1 converting enzyme;
PARP, poly(ADP-ribose) polymerase;
TLCK, N-
-p-tosyl-L-lysine
chloromethyl ketone;
U1-70K, 70-kD protein component of U1 small nuclear ribonucleoprotein.