1. Art and Diseño

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Protease Inhibitors Differentially Regulate Tumor Necrosis Factor-Induced
Apoptosis, Nuclear Factor-NB Activation, Cytotoxicity, and Differentiation
By Masahiro Higuchi, Sanjaya Singh, Henry Chan, and Bharat B. Aggarwal
We investigatedthe effect of various proteaseinhibitors on
TPCK-sensitive proteases are
not involved in the early stages
several tumor necrosisfactor (TNF)-mediated cellularreof signal transduction. TNF is cytotoxic
and induces differensponses. Treatment of a human myelogenous leukemia cell tiation in ML-la cells after a 3-day incubation.TPCK had no
line, ML-la, withTNF in the presence of cycloheximidetrigeffect on the TNF-induced cytotoxicity and differentiation,
gers endonucleolytic
activity and apoptotic celldeath within
indicatingthat TPCK-sensitive proteases are specific for DNA
90 minutes. The general serine protease inhibitor diisopropyl
fragmentation. TPCKalso blocked TNF-induced activation
fluorophosphate (DFP) and the chymotrypsin-like protease
of nuclear factor (NFI-KB. The dose-response andthe timeinhibitorN-tosyl-L-lysylchloromethyl ketone (TPCK)comcourse of the inhibitor, however, indicated that the site of
pletely abrogated TNF-induced DNA fragmentation and the
action ofTPCK for NF-KB activationand for DNA fragmentaformation of apoptotic bodies. However, 13 other protease
tion are quite distinct. Therefore,we conclude that TNF actiinhibitors, including serine protease inhibitors,
did not. The
to
vates two distinct TPCK-sensitive pathways, one leading
addition ofTPCK to cells 30 minutes after TNF treatment
apoptosis and the other to NF-KB activation.
completely inhibited the cytokineaction,indicating
that
0 1995 by The American Society of Hematology.
T
UMOR NECROSIS FACTOR (TNF) was first characterized from the sera of rodents infected with Bacillus
Culmette-Guerin and subsequently treated with endotoxin;
it was thencharacterized as a monokine that induces necrosis
of certain tumors in vivo.’ Since then, a wide variety of
biologic activities in vitro have been ascribed to TNF, including antiproliferative activity against tumor cells, stimulation
of human fibroblasts, B-cell and thymocyte proliferation,
induction of differentiation, antiviral effects, and induction
of various gene expression.’ TNF now is known to beone of
the most important mediators of cytotoxicity, inflammation,
autoimmune diseases, and bone resorption in vivo.’
TNF interacts with a wide variety of cell types that express
specific cell-surface receptors. The cDNA for two types of
TNF receptor with approximate molecular masses of 60 kD
and 80 k D , respectively, have been isolated.”6 Several reports showed that the signal through the p60 receptor is
necessary for most biologic functions of TNF including cytotoxicity, the growth stimulation of fibroblast cells, induction
of Mn-superoxide dismutase, antiviral activity, and endotoxin shock in
However, p80 receptor is also known
to mediate cytotoxicity, proliferation of murine thymocytes
and humanmononuclear cells, and induction of granulocytemacrophage colony-stimulating factor (GM-CSF) secretion.10.’3-ih
From the Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, m.
Submitted February 7, 1995; accepted May 10, 1995.
This research was conducted in part by The Clayton Foundation
for Research and was supported in part by new program development funds from the University of Texas M.D. Anderson Cancer
Center.
Address reprint requests to Bharat B. Agganual, PhD, Cytokine
Research Laboratory. Department of Molecular Oncology, BOX41,
The University ofTexas M.D. Anderson Cancer Center, Houston,
TX 77030.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8606-0012$3.00/0
2248
We have shown that a myelogenous leukemia cell line,
ML-la, exhibits cytotoxicity and differentiation in the presence of 10% fetal calf serum (FCS) after a 3-day incubation
with TNF and apoptotic cell death within 4 hoursinthe
absence of FCS.17Although signal mediated through the p60
receptor is sufficient to induce apoptosis, signals mediated
through both receptors are needed to induce differentiation.I7
The postreceptor signals needed for TNF-mediated
apoptosis, cytotoxicity, and differentiation, however, have
not yet been identified.
A number of hypotheses have been postulated to explain
the mechanisms by which TNF cytotoxicity occurs. Signaling intermediates including oxygen radicals,’”-’’ phospholipase A2 (PLA2) activation,”~’5 sphingomyelinase activation,”and adenosine diphosphate (ADP) r i b o ~ y l a t i o n ~ ~ ~ ’ ~
have been implicated in the TNF action. As protease inhibitors suppress TNF-mediated cytotoxicity, a role for proteases
has also been
As
de novo protein synthesis is
not needed for TNF cytotoxicity,” protease activation rather
than new synthesis may be required.
We recently developed a bioassay system in which human
TNF induces apoptotic cell death in ML- 1 a cells in the presence of cycloheximide within 90 minute~.~’
In the present
study, we used this system to investigate the mechanism of
TNF-induced apoptosis in ML-la cells. We report a specific
protease inhibitor-sensitive pathway leading to apoptosis by
TNF that is distinct fromthat of the pathwayleadingto
nuclear factor (NF)-KB activation.
MATERIALS AND METHODS
Materials. Gentamicin, RPMI-1640 medium, and FCS were obNY). DiisopropylfluorophostainedfromGIBCO(GrandIsland,
phate (DFP), phenylmethanesulfonyl fluoride (PMSF). aprotinin, Ntosyl-L-phenylalanyl chloromethyl ketone (TPCK), N-tosyl-L-lysyl
Echloromethyl ketone (TLCK), benzamidine, leupeptin, antipain,
64, acetyl-leu-Leu nonnethional, pepstatin, phosphoramidon, captopril, N-tosyl-L-arginine methyl ester (TAME), dimethyl sulfoxide,
NaCI, RNase K, proteinase K, bromphenolblue,xylenecyano],
glycerol, okadaic acid, H-7, and nitroblue tetrazolium were obtained
MO). The Bowman-Birk inhibifrom Sigma Chemical CO (St Louis,
from Dr Ann Kennedy of the University of
tor (BBI) was a gift
Pennsylvania (Philadelphia, PA). Bacteria-derived recombinant human TNF, purified to homogeneity with a specific activity of 5 X
IO’ Ulmg,wasprovided by Genentech Inc (SouthSan Francisco.
CA).
Blood, Vol 86,No 6 (September 15). 1995: pp 2248-2256
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2249
REGULATION OF TNF-INDUCED SIGNALTRANSDUCTION
Cell line. Myelogenous leukemia ML-la cell line was obtained
from Dr Ken Takeda (Showa University, Tokyo, Japan) and was
grown in RPMI-1640 medium supplemented with 10% FCS and
gentamicin (50 pg/mL) (essential medium). The cells were seeded
at a density of 1 X l@/mL in T-25 flasks (Falcon 3013, Becton
Dickinson Labware, Lincoln Park, NJ) containing 10 mL of essential
medium and were grown at 37°C in an atmosphere of 95% air and
5% CO,. Cell cultures were split every 3 or 4 days.
Cytotoxicityassay. The cytotoxicity of ML-la cells was estimated by the inhibition of I3H]TdR incorporation into ML-la cells.
For this, cells (5 X 103/well)were incubated with TNF for 72 hours.
During the last 6 hours, ['HITdR was added to each well (0.5 pCi
per well). Then, the cell suspension was harvested with the aid
of a Filtermate 196 (Packard Instrument CO Ltd, Meriden, CT).
Radioactivity bound to the filter was measured by Matrix 9600
(Packard). Cytotoxic activity was calculated as follows: % cytotoxicin control sample)]
ity = [ l - (t3H]TdR in test ~ample)/([~H]TdR
X 100. All results were determined in triplicate and expressed as
mean 2 standard error. The results from this method of examining
cytotoxicity correlated with that obtained by 3-(4-5-dimethylthiozol2-yl) 2-5-diphenyl-tetrazolium bromide ( M m ) assay or trypan blue
exclusion method.
DNA fragmentation assay by ['HITdR release. DNA fragmentation induced by TNF was assayed by the modified method as described." Briefly, ML-la cells were prelabeled with [3H]TdR by
incubating 2 X lo5 cells per milliliter in essential medium with 0.5
pCi/mI. t3H]TdR at 37°C for 16 hours. Then, the cells were washed
three times and resuspended in RPMI-1640 medium and plated in
96-well plates (4 X lo4 per well; total volume, 200 pL) with or
without the test samples. To potentiate the TNF response, 1 mg/mL
of cycloheximide was in~luded.3~
After incubation for 90 minutes
or the indicated time, they were lysed by the addition of 50 pL of
detergent buffer (10 mmoVL Tris-HCI, pH 8.0, containing 5 mmoV
L EDTA and 2.5% Triton X-100) and incubated an additional 15
minutes at 4°C. High-speed centrifugation was performed on an
Eppendorf microcentrifuge at 12,OOOg for 1 minute. The radioactivity in the supernatant represents DNA release into cells due to DNA
fragmentation. For the total count, the cells were lysed by the addition of 20 pL of 20% sodium dodecyl sulfate (SDS). The percent
DNA release was calculated as follows: 76 DNA fragmentation =
(cpm in test sample supernatantkotal cpm) X 100. Each assay was
performed in triplicate, and results are shown as the mean t standard
error.
Analysis of DNA fragmentation by agarose gel. After treatment
of ML-la cells (1 X 106/0.2mI.) with TNF, cells were centrifuged,
washed with phosphate-buffered saline, and resuspended in 40 pL
of the lysis buffer (10 mmol/L Tris-Cl, pH 8.0, 100 mmom NaCI,
25 mmolk EDTA, and 0.5% SDS) containing 20 pg of RNase A.
After incubation at 50°C for 30 minutes, 1 pL of 20 mg/mL proteinase K was added to the reaction mixture, and incubation was continued for another 30 minutes. Next, 8 pL of 6X loading dye (0.025%
bromophenol blue, 0.25% xylene cyano1 FF, and 30% glycerol in
water) was added, and 40 pL of the sample was resolved on 1.2%
agarose gel in TAE buffer (0.04 molk Tris-acetate, 0.001 molk
EDTA).
Differentiation assay. Differentiation-inducing activity was assayed colorimetrically by the modified method of Baehner and Nathan.34Briefly, 4 X lo4 cells were plated in 96-well plates in the
presence of test samples at a final volume of 200 pL and incubated
for 3 days. Then, 20 pL of PBS containing 1.1 mg/mL of nitroblue
tetrazolium and 1.1 pmolk phorbol myristate acetate (PMA) was
added, and the solution was incubated for another 2 hours. The
reaction was then terminated by adding 50 pL 2 N HCI and cooling
on ice for 30 minutes. The medium was discarded, the formazan
deposits were dissolved by adding 0.1 mL dimethyl sulfoxide, and
the dissolved formazan was measured at 590 nm by a spectrophotometer. The final results were calculated and expressed as absorbance at 590 nm/106 cells. Each assay was performed in triplicate,
and results are shown as the mean ? standard error.
NF-KB induction assays. ML-la cells (2 X 106/0.1 mL) were
treated with or without 0.1 nmolk TNF in the presence or absence
of the indicated amount of TPCK for the indicated time at 37°C.
Nuclear extracts were prepared according to Schreiber et al.35Briefly,
2 X lo6 cells were washed with cold PBS and suspended in 0.4 mL
of lysis buffer (10 mmolk HEPES pH 7.9, 10 mmolk KCl, 0.1
mmolk EDTA, 0.1 mmoVL EGTA, 1 mmolk dithiothreitol [Dm],
0.5 mmom PMSF, 2.0 p g h L leupeptin, 2.0 p@mL aprotinin, and
0.5 mg/mL benzamidine). The cells were allowed to swell on ice
for 15 minutes; after which time, 25 pL of 10% NP-40 was added.
The tube was then vigorously vortexed for 10 seconds. The homogenate was centrifuged for 30 seconds in a microfuge. The nuclear
pellet was resuspended in 25 pL ice-cold nuclear extraction buffer
(20 mmolk HEPES pH 7.9, 0.4 mmoVL NaC1, 1 mmolk EDTA,
1 mmolk EGTA, 1 mmoVL DTT, 1 mmolk PMSF, 2.0 p@mL
leupeptin, 2.0 pg/mL aprotinin, and 0.5 mg/mL benzamidine), and
the tube was incubated on ice for 15 minutes with intermittent mixing. This nuclear extract was then centrifuged for 5 minutes in a
microfuge at 4°C. and the supernatant was frozen at -70°C. The
protein content was measured by the method of Bradford.36
Electrophoretic mobility shift assays were performed by incubating 4 to S pg of nuclear extract with 12 fmol of 32P-endlabeled,
45-mer double-stranded, NF-KB oligonucleotide from human immunodeficiency virus- 1 long terminal repeat (5"TTGTTACAAGGGACT'ITCCGCTGGGGACTlTCCAGGGAGGCGTGG-3')37in the
presence of 0.5 pg of poly (dl-dC) in a binding buffer (25 mmolk
HEPES pH 7.9, 0.5 mmoVL EDTA, 0.5 mmolk DTT, 1% NP-40,
5% glycerol, and 50 mmolk NaC1)3K.39
for 20 minutes at 37°C. The
DNA-protein complex formed was separated from free oligonucleotide on a S% native polyacrylamide gel using buffer containing 50
mmom Tris, 200 mmolk glycine pH 8.5, and 1 rnmoVL EDTA."9
The gel was fixed in 10% acetic acid and dried. Quantitation and
visualization of radioactive bands were performed using a phosphorimager (Molecular Dynamics, Sunnyvale, CA) with Imagequant software.
RESULTS
Effect of protease inhibitors on TNF-induced apoptosis.
A number of protease inhibitors were tested for their effects
on TNF-induced DNA fragmentation. The concentration of
inhibitors used was not toxic as estimated by trypan blue
method but was known to inhibit the appropriate protease
action. The dose of each inhibitor required to inhibit 50%
DNA fragmentation induced by 1 nmoVL TNF is shown in
Table 1. The serine protease inhibitor DFP and chymotrypsin
inhibitor TPCK showed a potent ability to prevent TNFinduced DNA fragmentation. TPCK was approximately lo4
times more potent in inhibiting TNF-induced DNA fragmentation than DFF' (Fig 1). However, other serine protease
inhibitors such as PMSF, aprotinin, TLCK, BBI, and other
inhibitors including leupeptin, antipain, E-64, acetyl-LeuLeu normethional, pepstatin, phosphoramidon, captopril, and
TAME did not inhibit TNF-induced DNA fragmentation at
the doses tested, indicating that chymotrypsin-like serine
protease may be involved in TW-induced DNA fragmentation. We also determined the effect of TPCK on TNF-induced DNA fragmentation using agarose gel (Fig 2). The
degradation of intact DNA and the formation of ladder was
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HlGUCHl ET AL
2250
Table 1. The Effect of Protease Inhibitors onTNF-Induced
Apoptosis
Agent
DFP
PMSF
TPCK
BTEE
APNE
BB1
TLCK
Leupeptin
Antipain
E-64
Acetyl-Leu-Leu
normethional
Pepstatin
Phosphoramidon
Captopril
TAME
ID-
Specificity
4.5 mmol/L
>5 mmol/L
Serine protease
Serine protease
Chymotrypsin
chymotrypsin
Chymotrypsin
Trypsin-chymotrypsin
Trypsin
Trypsin, cysteine protease
Trypsin, cysteine protease
Cysteine protease
0.22 pmol/L
35 pmol/L
38 pmol/L
>l00 pg/mL
>300 pmol/L
>ZOO pmol/L
>l00 pmol/L
>l00 pmol/L
Calpain II
>l00 pmol/L
Some aspartic protease
Metalloprotease
Angiotensin converting
enzyme
Nonspecific
> l 0 0 pmol/L
>l00 pmol/L
>2 mmol/L
>5 mmol/L
l3H1TdR-prelabeled ML-la cells (4 x 10') were incubated with 1
nmol/L TNF and 1 pg/mL cycloheximide in the presence or absence
of the indicated amounts of protease inhibitors for 90 minutes and
then were tested for DNA fragmentation as described in Materials
and Methods. The amounts of protease inhibitors that showed a 50%
inhibition of DNA fragmentation are shown.
Abbreviations: BTEE, N-benzoyl-L-tyrosine ethyl ester;APNE, Nacetyl-DL-phenylalanine o-naphthvl ester.
Concentration
(uM)
Fig 1. Inhibition ofTNF-induced DNA fragmentation byserine protease inhibitors. 13H1TdR-prelabeledML-la cells (4 x 10'1 were incubated with 1 nmol/LTNF and 1pg/mL cycloheximide in the presence
or absence of the indicated amountsof TPCK or DFP for 90 minutes
and were tested for DNA fragmentation as described in Materials
and Methods. The TPCK- and DFP-induced inhibitory levels were normalized to theamount of fragmented DNA in cells treated onlywith
TNF and cycloheximide.
Fig 2. Agarose gel electrophoresis of DNA fragments from TNFtreated cells. ML-la cells (2 x 10') were incubated in thepresence or
absence of TNF (1 nmol/L) and cycloheximide (l pg/mL) with or
without TPCK (10 pmol/L) for 90 minutes, and DNA from cells was
processed for electrophoresis as described in Materials and Methods.
observed in the cells treated with TNF and cycloheximide.
and TPCK completely blocked this effect. We further investigated the effect of TPCK on the morphologic changes of
ML-la cells after TNF treatment. A 90-minute TNF treatment in the presence of cycloheximide induced theformation
of apoptotic bodies (Fig 3). and this change was abrogated
by the addition of TPCK, indicating thatTPCKblocked
TNF-induced apoptotic body formation. There was no detectable formation of apoptotic bodies in the control cells or
in the TPCK-treated cells. DFP also completely abrogated
the apoptotic body formation in ML-la cells induced by
TNF (data not shown). These results clearly demonstrate that
TPCK blocked TNF-induced apoptosis in ML- I a cells.
Kinetics o f inhihitiorl of TNF-induced DNA,frcr~lnentfition
/?yTPCK. To determine where the TPCK-sensitive element
resides in the pathway leading to apoptosis induced by TNF,
TPCK was added at the same time as or 30 or 60 minutes
after TNF addition, andinhibition ofDNA fragmentation
was measured (Fig 4). The addition of TPCK at time 0 and
30 minutes completely blocked the TNF effect. When TPCK
waspresentonly in the last 30 minutes of the 90-minute
reaction time. DNA fragmentation was significantly but partially inhibited. indicating that this TPCK-sensitive component was activated from 30 to 90 minutes after TNF treatmen t .
Efect o f TPCK on H-7- nnd okodoic acid-induced DNA
.frapentntiorr. We also examinedwhetherTPCKinhibits
apoptosis induced by inducers other than TNF. As protein
kinase C inhibitor H-7 and serinekhreonine protein phosphatase inhibitor okadaic acid"'." have also beenreportedto
induce apoptosis. we examined the effect of TPCK on
apoptosis induced by these agents. ML-la cells were incubated with H-7 or okadaic acid for 4 hours, and the effect
of TPCK was examined. As shown in Fig S , TPCK nearly
completely inhibited H-7- and okadaic acid-induced DNA
fragmentation. thus suggesting that TPCK is a more general
inhibitor of apoptosis.
Efect of TPCK on TNF-induced cytoto.ricify nnd diferentintion. TPCK may interfere withanearlyandgeneral
event of TNF signals that is linked to varieties of biologic
functions, or it may block just the apoptosis-specific signals.
To examine this, we measured the effects of TPCK on two
different biologic functions: cytotoxicity and differentiation
in ML-la cells. Treatment with 1 pglmL ofTPCK com-
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REGULATION OF TNF-INDUCED SIGNAL TRANSDUCTION
2251
+TPCK
?
+TNF
I
l
Fig 3. The effect of TPCK onthemorphologic
changes induced by TNF in ML-la cells. Cells were
incubated in the absence or presence of TNF (1nmoll
L) and cycloheximide (1 pg/mL) with and without
TPCK ( l 0 p m o l / L I for 90 minutes, and then micrographs weretaken(originalmagnification
x 2001.
I
TPCK treatment
'
,
1
-'
.
T
T
0 None
1
W +TPCK
0
10
100
loo0
TNF (PM)
Fig 4. Effect of TPCK added atvarious times afterTNF treatment.
['HlTdR-prelabeled ML-la cells (4 x 10') were incubatedwith 1nmoll
added
LTNF and1p g l m L cycloheximide. TPCK (10 pmol/L) was then
at 0, 30, and 60 minutes after TNF addition. At time 90 minutes,
the reaction wasstopped, and fragmented DNA was determined as
described in Materials and Methods.
control
H-7
okadaic acid
Fig 5. Inhibition ofH-7- and okadaic acid-induced DNA fragmentation by TPCK. ['HlTdR-prelabeled ML-la cells (4 x 10') were incubated with either 500 pmol/L H-7 or 500 pmollL okadaic acid in the
presence or absence of 10 pmol/L TPCK for 4 hours and then were
tested forDNA fragmentation as described in Materials andMethods.
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HlGUCHl ET AL
10'
102
lo3
Fig 6. The effect of TPCK on TNF-induced DNA fragmentation, cytotoxicity, and differentiation.(A) [3H]TdR-prelabeledML-la cells (4 x 10')
were incubated with the indicated amount of TNF and 1 pglmL cycloheximide in the presence or absenceof 1 pmollL TPCK for 90 minutes
and were tested for DNA fragmentation as described in Materials and Methods. (B) ML-la cells (5 x l@)were incubated with the indicated
amount of TNF in the presence or absence of 1 pmollL TPCK for 3 days and were tested for their cytotoxic activity as described in Materials
and Methods. (C) Cells (4 x 10') were incubated with the indicated amount of TNF and in the presence or absence of 1 pmollL TPCK for 3
days and were tested for their differentiation-inducing activityas described in Materials and Methods.
pletely blocked TNF-induced DNA fragmentation (Fig 6A),
but the same dose did not affect the cytotoxicity (Fig 6B)
and differentiation-inducing effect (Fig 6C) of TNF. The
results clearly indicate that the specific blockade of
apoptosis-specific signaling of TNF is independent of cytotoxicity or differentiation. Due to the toxicity of the inhibitor
alone, we could not use TPCK at concentration higher than
5 pmol/L. (data not shown).
Effect of TPCK on the induction of NF-KB. Similar to
apoptosis, another early action of TNF is activation of NFKB. The NF-KB activation by TNF in ML-la cells can be
noted within 15 minutes, and its level remained unchanged
until up to 90 minutes (Fig 7). Furthermore, cycloheximide
had no effect on the TNF-dependent NF-KB activation. We
also investigated whether TPCK inhibited TNF-induced NFKB activation. As shown in Fig 8A, TPCK abrogated rapid
induction of NF-KB by TNF in ML-la cells. However the
component for NF-KB activation was approximately 50
times more resistant to TPCK than that for the induction of
DNA fragmentation (Fig 8B).
DISCUSSION
Our results presented here demonstrate that a TPCK- and
DFP-sensitive element is involved in the signal transduction
sequence of TNF-induced apoptosis. This element is specific
to apoptosis but was not involved in TNF-induced cytotoxic-
ity and differentiation. We also found a TPCK-sensitive element in the NF-KB activation pathway, but this element
was completely distinct from that in the pathway leading to
apoptosis: the former appears much earlier (less than 15
minutes) and requires a 50-times higher dose of TPCK.
We have previously shown that the trypsin-type protease
inhibitor TLCK can inhibit the cytotoxic action of mouse
TNF in L.P3 cells, a clone of a mouse fibroblast cell line L929.29Others have also reported that serine protease inhibitors abrogated the effect of human TNF on L-929 ~ e l l s . ~ ' , ~ '
These experiments used human TNF on murine target cells.
Both p60 and p80 forms of TNF receptors are capable of
transducing the ~ignal.'~."~''
Because human TNF binds only
the murine p60 TNF receptor, human target cells that interact
with both receptors are preferable.
Most of the human TNF assay systems currently available
to study signaling require a 2- to 3-day incubation period,
which is undesirable because, during this time, inhibitors
themselves maybetoxictothe
cells. To investigate the
mechanisms of action of human TNF, we developed a system
to detect TNF-induced DNA fragmentation in ML-la, which
requires 90 minutes of incubation time. We demonstrated
that the protease inhibitors TPCK and DFP blocked TNFinduced apoptosis. TPCK is an alkylating agent, reacting
specifically and irreversibly with the histidine residue in the
active center of proteases, and has a high affinity for chymo-
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2253
REGULATION OF TNF-INDUCEDSIGNALTRANSDUCTION
0
1
5
60 30
90
time ( m i d
NF-KB
B
Fig 7. Kinetics of NF-KB activation by TNF. ML-la cells (2 x 10')
were incubated with 1 nmol/L TNF in the presence or absence of
1 p g l m L cycloheximide (CHXI for the indicated times, and NF-KB
activation was estimatedas described in Materials and Methods.
100
r
NF-kB
activation
80
trypsin andchymotrypsin-like enzymes."' DFP is anirreversible serine protease inhibitor reacting with serine residues in
its active center. Both are hydrophobic low-molecularweight molecules and are expected to penetrate the cell membrane and act intracellularly. In contrast, serine protease inhibitors aprotinin and BBI, which do not penetrate the cells
and are thus supposed to act extracellularly, did not affect
TNF action. Therefore, TNF may activate an intracellular
chymotrypsin-like serine protease or induce the sensitivity
of the substrate to the TPCK-sensitive protease. Although
TPCK is also known to inhibit protein synthesis: the effect
of TPCK onTNF action is notlikely to be due to thesuppression of protein synthesis, because we used protein synthesis
inhibitor cycloheximide in our assay system. Interestingly,
in our studies, the protease inhibitors (PMSF and TLCK)
that are known to block TNF-mediated cytotoxicity,'"-3' had
no effect on TNF-mediated apoptosis in ML-la cells; thus,
again suggesting that the pathway leading to apoptosis is
distinct from that of cytotoxicity. This TPCK-sensitive elementisnot
specific to TNF-induced apoptosis, as TPCK
also inhibited the apoptosis induced by H-7 and by okadaic
acid.
The molecular mechanisms associated with the apoptotic
process are not entirely clear. TNF has beenreportedto
induce apoptosis characterized by membrane blebbing, chromatin margination, and the breakdown of chromosomal
DNA into nucleosome-sized fragments in some tumor
cells.*~JsIn our previous studies, we also noted apoptotic
60
40
20
(I
l
10"
loo
10'
TPCK (uM)
Fig 8. Inhibition of TNF-induced NF-KB activation by TPCK. (AI
ML-la cells (2 x 10') were incubated with or without1 nmoi/L TNF
in the presence of the indicated amount of TPCK for 90 minutes,
and NF-KB induction was estimated as described in Materials and
Methods. (B) The effect of TPCK on NF-KB activation wascompared
with DNA fragmentation, both induced by TNF. The TPCK-induced
inhibitory levels were normalized t o the fragmented DNA and the
amount of activated NF-KB in cells treated only with TNF.
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2254
body formation, nucleosome-sized fragments of DNA on
agarose gels, and DNA fragmentation resulting in thymidine
release under identical conditions after TNF treatment of
ML-la cells.’7 Whether TNF induces apoptosis by the same
mechanisms as in other systems has notbeen answered.
However, some characteristics of TNF-induced apoptosis in
ML-la cells are different from T-cell apoptosis induced by
agents such as anti-CD3 antibodies. Apoptosis in T cells
requires new protein synthesis,* but the TNF-induced process is not inhibited by a protein synthesis inhibitors,32suggesting that these cells already have the molecules required
for this process. T-cell receptor-triggered programmed cell
death was inhibited by the cysteine protease inhibitor E-64
and leupeptin, the calpain-selective inhibitor acetyl-Leu-Leu
normethional, and the serine protease inhibitors DFP and
PMSF.47Like TNF, topoisomerase I is another inducer of
apoptosis that does not require new protein s y n t h e ~ i sthis
;~~
apoptosis, however, is blocked notonly by TPCK but also by
other serine protease inhibitors including TLCK and TAME.
From the difference of spectrum of protease inhibitors, we
conclude that distinct proteases are involved in apoptosis of
T cells, in topoisomerase I-induced apoptosis, and in TNFinduced apoptosis.
Two genes, ced-3 and ced-4, are essential for cells to
undergo programmed cell death in C e l e g ~ n sThe
. ~ ~amino
acid sequence of CED-3 protein is similar to the mammalian
interleukin (1L)-l@-converting enzyme.50This protein is a
cysteine protease, and overexpression of this gene causes
the cells to undergo a p o p t o ~ i sHowever,
.~~
the proteases activated by TNF are clearly different from the IL-l@-converting enzyme because of the differences in the spectrum
of action of protease inhibitors.
The role of ADP ribosylation in TNF-mediated cytotoxicity has been dem~nstrated.~’.~’
It was also proposed that the
proteolytic cleavage of the poly (ADP-ribose) polymerase,
a 116-kD protein, may be involved in apoptosis, because
cleavage to 85- and 25-kD fragments and apoptosis induced
by etoposide were shown to be sim~ltaneous.~’
At present.
we do not know whether proteases involved in this proteolytic cleavage and those in TNF-induced apoptosis are the
same. Collectively, several distinct proteases may have the
potential to induce apoptosis.
Are different mechanisms involved in TNF-mediated cytotoxicity in L.P3 cells and L-929 cells, apoptotic cell death
in ML- la cells, and cytotoxicity in ML-la cells? Cytotoxic
activity that can be observed 3 days after TNF treatment in
ML- la cells may be mainly the results of the cytostatic action
of TNF. Mitochondrial dysfunction can decrease intracellular adenosine triphosphate (ATP) levels, and this may lead
to cytostasis but not ~ y t o l y s i sSince
. ~ ~ TNF has been shown
to induce damage in the mitochondrial respiratory chain in
TNF-sensitive cell^,'^^^^ this mechanism may also be responsible for cytotoxicity in ML-la cells. TNF-induced cytotoxicity in ML- 1a cells was not inhibited by TPCK, suggesting
that TPCK-sensitive protease is not involved in the dysfunction of mitochondrial respiratory chain. In L.P3 and L-929
cells, TNF induces cytolysis (membrane disruption) without
DNA fragmentati~n.~~
We and others have demonstrated that
the TNF-mediated damage to the mitochondrial respiratory
HlGUCHl ET AL
chain precedes the PLA2 activation, leading to lysis of L.P3
and L-929
Serine proteases that act in L.P3
and
L-929 cells after TNF treatment may transmit the signals
from mitochondria disfunction, leading to PLA2 activation.
Apoptotic cell death was rapidly induced after TNF and
cycloheximide treatment without membrane disruption.”
Whether mitochondrial damage is essential to induce
apoptosis, however, needs to be examined. At the least, distinct mechanisms may be involved in these three cytotoxic
mechanisms induced by TNF.
TNF regulates the transcription of several genes, and some
of them are regulated by the transcriptional factor N F - K B . ~ ~
The activation of NF-KB involves the dissociation of the
heterodimeric components p65 and p50 from the inhibitory
subunit IKB, followed by its translocation to the nucleus.55
Rapid degradation of IkB was found to be a key event for this
mechanism.56Machleidt et a157reported that serine protease
inhibitors blocked the degradation of I k B and, thus, inhibited
NF-KB activation. Our results show that in the signal transduction of TNF, the TPCK-sensitive component involved in
apoptosis and that involved in NF-KB activation were quite
different for the following reasons. (1) The TPCK-sensitive
component for apoptosis appeared later. (2) The element for
NF-KBactivation was approximately 50 times more resistent
to TPCK than that for DNA fragmentation. Although these
two pathways are distinct, we could not estimate whether
they are linked in some manner. Because of the difference
in conditions required, we could not examine whether the
TPCK ( l 0 pmoVL)-sensitivepathway involved in NF-KB
activation (30 minutes) is also involved in cytotoxicity and
differentiation (72 hours). Future studies on the characterization of the TPCK-sensitive proteases may provide further
insight into the mechanisms of TNF action.
ACKNOWLEDGMENT
We are verythankful to DrBryant G. Darnay,DrShrikanth
Reddy, and Walter Pagel for their thoughtful reviews of the manuscript and helpful suggestions.
REFERENCES
I , Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson
B: An endotoxin-induced serumfactorthat causes necrosis of tumors. Proc Natl Acad Sci USA 72:3666, 1975
2. Aggarwal BB, Vilcek J (eds): Tumor Necrosis Factor: Structure, Function, and Mechanism of Action. New York, NY, Marcel
Dekker, 1992, p 1
3. Loetscher H, Pan Y-CE, Lahm H-W, Gentz R, Brockhaus M,
Tabuchi H, Lesslauer W: Molecular cloning and expression of the
human 55 kd tumor necrosis factor receptor. Cell 61:351, 1990
4. Schall TJ, Lewis M, Koller KJ, Lee A, Rice G C , Wong GHW,
Gatanaga T, Granger GA, Lentz, R, Raab H, Kohr WJ, Goeddel
DV: Molecular cloning and expression of areceptorforhuman
tumor necrosis factor. Cell 61:361, 1990
5. Smith CA,Davis T, Anderson D, Solam L, Beckman MP,
Jerzy R, Dower SK, Cosman D, Goodwin RG: A receptor for tumor
necrosis factor defines an unusual family of cellular and viral proteins. Science 248:1019, 1990
6 . Kohno T, Brewer MT, Baker SL, Schwaltz PE, King MW,
Hale KK, Squires CH, Thompson RC, Vannice JL: Second tumor
necrosis factor receptor gene product can shed a naturally occumng
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
REGULATION OF TNF-INDUCEDSIGNALTRANSDUCTION
tumor necrosis factor inhibitor. Proc Natl Acad Sci USA 87:8331,
1990
7. Espevik T, Brockhaus M, Loetscher H, Nostad U, Shalaby R.
Characterization of binding and biological effects of monoclonal
antibodies against a human tumor necrosis factor receptor. J Exp
Med 171:415, 1990
8. Englemann H, Holtman H, Brakebusch C, Avin, YS, Sarov I,
Nophar Y, Hadas E, Leitner 0, Wallach D: Antibodies to a soluble
form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem 265:14497, 1990
9. Thoma B, Grell M, Plizenmaier K, Scheurich P: Identification
of 60-kD tumor necrosis factor (TNF) receptor as the major signal
transducing component in TNF responses. J Exp Med 172:1019,
1990
IO. Tartaglia LA, Weber RF, Figari IS, Reynold C, Palladino MA
Jr, Goeddel DV: The two different receptors for tumor necrosis
factor mediate distinct cellular responses. Proc Natl Acad Sci USA
88:9292, 1991
11. Wong GHW, Tartaglia LA, Lee MS, Goeddel DV: Antiviral
activity of tumor necrosis factor (TNF) is signaled through the 55kDa receptor, type I TNF. J Immunol 149:3350, 1992
12. Pfeffer K, Matsuyama T, Kundig TM, Wakeham A, Kishihara
K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, Mak TW:
Mice deficient for the 55 kd tumor necrosis factor receptor are resistent to endotoxin shock, yet succumb to L. monocytogenes infection.
Cell 73:457, 1993
13. Grell M, Scheurich P, Meager A, Pfizenmaier K: TR60 and
TR80 tumor necrosis factor (TNF)-receptors can independently mediate cytolysis. Lymphokine Cytokine Res 12:143, 1993
14. Heller RA, Song K, Fan N, Chang DJ: The p70 tumor necrosis
factor receptor mediates cytotoxicity. Cell 70:47, 1992
15. Gehr G , Gentz R, Brockhaus M, Loetscher H, Lesslauer W:
Both tumor necrosis factor receptor types mediate proliferative signals in human mononuclear cell activation. J Immunol 149:911,
1992
16. Vandenabeele P, Declercq W, Vercammen D, Van de Craen
M, Grooten J, Loetscher H, Brockhaus M, Lesslauer W, Fiers W:
Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma. J Exp Med
176:1015, 1992
17. Higuchi M, Aggarwal BB: Differential roles of two forms of
TNF receptor on TNF-induced cytotoxicity, DNA fragmentation and
differentiation. J Immunol 152:4017, 1994
18. Matthews N, Neale ML, Jackson SK, Stark JM: Tumor cell
killing by tumor necrosis factor: Inhibition by anaerobic conditions,
free-radical scavengers and inhibitors of arachidonate metabolism.
Immunology 62:153, 1987
19. Higuchi M, Shirotani K, Higashi N. Toyoshima S, Osawa T:
Damage to mitochondrial respiration chain is related to phospholipase AZ activation caused by tumor necrosis factor. J Immunother
2:41, 1992
20. Schulze-Osthof K, Bakker AC, Vanhaesebroeck B, Beyaert
R, Jacob WA, Fiers W: Cytotoxic activity of tumor necrosis factor
is mediated by early damage of mitochondrial functions. J Biol Chem
267:5317, 1992
21. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman
G , Fiers W: Depletion of mitochondrial electron transport abrogates
the cytotoxic and gene-inductive effects of TNF. EMBO J 12:3095,
1993
22. Kobayashi Y, Sawada J, Osawa T Activation of membrane
phospholipase A by guinea pig lymphotoxin (GLT). J Immunol
122:791, 1979
23. Neale ML, Fiera RA, Matthews D: Involvement ofphospholipase A2 activation in tumor cell killing by tumor necrosis factor.
Immunology 64:81, 1988
2255
24. Oka S, Arita H Inflammatory factors stimulate expression of
group I1 phospholipase Az in rat cultured astrocytes. Two distinct
pathways of the gene expression. J Biol Chem 266:9956, 1991
25. Hayakawa M, Ishida N, Takeuchi K, Shibamoto S, Hori T,
Oku N, It0 F, Tsujimoto M: Arachidonic acid-selective cytosolic
phospholipase A2 is crucial in the cytotoxic action of tumor necrosis
factor. J Biol Chem 268:11290, 1993
26. Kolesnick R, Golde DW: The sphingomyelin pathway in tumor necrosis factor and interleukin-l signaling. Cell 77:325, 1994
27. Aganval S, Drysdale B-E, Shin HS: Tumor necrosis factormediated cytotoxicity involves ADP-ribosylation. J Immunol
140:4187, 1988
28. Lichtenstein A, Gera JF, Andrew J, Berenson J, Ware CF:
Inhibitors of ADP-ribose polymerase decrease the resistance of
HERYneu-expressing cancer cells to the cytotoxic effects of tumor
necrosis factor. J Immunol 146:2052, 1991
29. Takeda Y, Shimada S, Sugimoto M, Woo HJ, Higuchi M,
Osawa T: Purification and characterization of a cytotoxic factor
produced by a mouse macrophage hybridoma. Cell Immunol96:277,
1985
30. Ruggiero V, Johnson SE, Baglioni C: Protection from tumor
necrosis factor cytotoxicity by protease inhibitors. Cell Immunol
107:317, 1987
31. Suffys P, Beyaert R, Van Roy F, Fiers W: Involvement of a
serine protease in tumor-necrosis-factor-mediated cytotoxicity. Eur
J Biochem 178:257, 1988
32. Kull FC, Cuatrecasas P: Possible requirement of internalizationin the mechanism ofin vitro cytotoxicity in tumor necrosis
serum. Cancer Res 41:4885, 1981
33. Higuchi M, Singh S, Aggarwal BB: Characterization of apoptotic effects of human tumor necrosis factor: Development of highly
rapid and specific bioassay for human tumor necrosis factor and
lympbotoxin using human target cells. J Immunol Methods 178:173,
1994
34. Baehner RL, Nathan DG: Quantitative nitroblue tetrazolium
test in chronic granulomatous disease. N Engl J Med 278:971, 1968
35. Schreiber E, Matthias P, Muller MM, Schaffner W: Rapid
detection of octamer binding proteins with ‘mini-extracts’, prepared
from a small number of cells. Nucleic Acids Res 175419, 1989
36. Bradford MM: A rapid and sensitive method for quantitation
of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248, 1976
37. Nabel G , Baltimore D: An inducible transcription factor activates expression of human immunodeficiencyvirus in T cells. Nature
326:7 1I, 1987
38. Collart MA, Baeuerle P, Vassalli P: Regulation of tumor
necrosis factor alpha transcription in macrophages: Involvement of
four KB-like motifs and of constitutive and inducible forms of NFKB. Mol Cell Biol 10:1498, 1990
39. Hassanain HH, Dai W, Gupta SL: Enhanced gel mobility shift
assay for DNA-binding factors. Anal Biochem 213:162, 1993
40. Mcconkey DJ, Hartzell P, Amardor-Perez JF: Orrenius S,
Jondal M: Calcium-dependent killing of immature thymocytes by
stimulation via the CD3m cell receptor complex. J Immunol
143:1801, 1989
41. Boe R, Gjertsen BT, Vintermyr OK, Houge G , Lanotte M,
Doskeland SO: The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian
cells. Exp Cell Res 195237, 1991
42. Schoellmann G , Shaw E: Direct evidence for the presence of
histidine in the active center of chymotrypsin. Biochemistry 2:252,
1962
43. Cbou I-N, Black PH, Rohlin RO: Non-selective inhibition of
transformed cell growth by protease inhibitor. Proc Natl Acad Sci
USA 71:1748, 1974
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2256
4 4 . Laster SM, Wood JG, Gooding LR: Tumor necrosis factor
can induce both apoptotic and necrotic forms of cell lysis. J Immunol
141:2629,1988
45. Wright SC, Kumar P, Tam AW, Shen N, Varma M, Larrick
JW: Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells. J Cell Biochem 48:344, 1992
46. Cohen JJ, Duke RC: Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J
Immunol 132:38, 1984
47. Sarin A, Adams DH, Henkart PA: Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a
murine T cell hybridoma and activated peripheral T cells. J Exp
Med 178:1693,1993
48. Bruno S, Bino GD, Lassota P, Giaretti W, Darzynkiewicz
Z: Inhibitors of proteases prevent endonucleolysis accompanying
apoptotic death of HL-60 leukemic cells andnormal thymocytes.
Leukemia 6: 1 113, 1992
49. Ellis H, Horvitz HR: Genetic control of programmed cell
death in the nematode C. elegans. Cell 44:817, 1986
50. Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J: Induction
of apoptosis in fibroblasts by IL-l@-converting enzyme, a mamma-
HlGUCHl ET AL
lian homolog of the C. elegans cell death gene ced-3. Cell 75:653.
1993
51. Kaufmann SH, Desnoyers S, Ottaviano Y, DavidsonNE,
Poirier GG: Specific proteolytic cleavage of poly(ADP4bose) polymerase: An earlymarker of chemotherapy-inducedapoptosis. Cancer
Res 53:3976,1993
52. Hibbs JB Jr, Taintor RR, Vavrin Z: Macrophage cytotoxicity:
Role for L-arginine deiminase and imino nitrogen oxidation to nitrite.
Science 235:473, 1987
53. Liddil JD, Dorr RT. Scuderi P: Association of lysosomal
activity with sensitivity and resistance to tumor necrosis factor in
murine L929 cells. Cancer Res 49:2722, 1989
54. Leonard MJ, Baltimore D: NF-KB: A pleiotropic mediator of
inducible and tissue-specific gene control. Cell 58:227, 1989
55. Baueurle PA, Baltimore D: I-KB: A specific inhibitor of the
NF-KB transcription factor. Science 242540, I988
56. Henkel T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah Y,
Baeuerle PA: Rapid proteolysis of I-KB-cxis necessary for activation
of transcription factor NF-KB. Nature 365:182, 1993
57. Machleidt T, WiegmannK, Henkel T, Schutze S, Baeuerle
P. Kronke M: Sphingomyelinase activates proteolytic IKB-cxdegradation in a cell-free system. J Biol Chern 269:13760, 1994
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 86: 2248-2256
Protease inhibitors differentially regulate tumor necrosis factorinduced apoptosis, nuclear factor-kappa B activation, cytotoxicity,
and differentiation
M Higuchi, S Singh, H Chan and BB Aggarwal
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