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Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells
by stabilization of death receptors
Matti Ahonen
1,2
, Minna Poukkula
1,3
, Andrew H Baker
4
, Masahide Kashiwagi
5
, Hideaki Nagase
5
,
John E Eriksson
1,3
and Veli-Matti Ka
¨ha
¨ri*
,
1,2
1
Centre for Biotechnology, University of Turku and A
˚bo Akademi University, FIN-20520 Turku, Finland;
2
Department of Medical
Biochemistry and Department of Dermatology, University of Turku, FIN-20520 Turku, Finland;
3
Department of Biology, University
of Turku, FIN-20014 Turku, Finland;
4
Department of Medicine and Therapeutics, University of Glasgow, Glasgow G11 6NT, UK;
5
Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London W6 8LH, UK
Tissue inhibitors of metalloproteinases (TIMPs) are
important regulators of matrix metalloproteinase
(MMP) and adamalysin (ADAM) activity. We have
previously shown that adenovirally expressed tissue
inhibitor of metalloproteinases-3 (TIMP-3) induces apop-
tosis in melanoma cells and inhibits growth of human
melanoma xenografts. Here, we have studied the role of
death receptors in apoptosis of melanoma cells induced by
TIMP-3. Our results show, that the exposure of three
metastatic melanoma cell lines (A2058, SK-Mel-5, and
WM-266-4) to recombinant TIMP-3, N-terminal MMP
inhibitory domain of TIMP-3, as well as to adenovirally
expressed TIMP-3 results in stabilization of tumor
necrosis factor receptor-1 (TNF-RI), FAS, and TNF-
related apoptosis inducing ligand receptor-1 (TRAIL-RI)
on melanoma cell surface and sensitizes these cells to
apoptosis induced by TNF-a, anti-Fas-antibody and
TRAIL. Stabilization of death receptors by TIMP-3
results in activation of caspase-8 and caspase-3, and
subsequent apoptosis is blocked by specific caspase-8
inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor
(Z-DEVD-FMK). Adenovirus-mediated expression of
TIMP-3 in human melanoma xenografts in vivo resulted
in increased immunostaining for TNF-RI, FAS, and
cleaved caspase-3, and in apoptosis of melanoma cells.
Taken together, these results show that TIMP-3 promotes
apoptosis in melanoma cells through stabilization of three
distinct death receptors and activation of their apoptotic
signaling cascade through caspase-8.
Oncogene (2003) 22, 2121–2134. doi:10.1038/sj.onc.1206292
Keywords: TIMP-3; death receptor; apoptosis; caspase;
melanoma
Introduction
Degradation of extracellular matrix (ECM) is instru-
mental in tumor growth, invasion, and angiogenesis
(Westermarck and Ka
¨ha
¨ri, 1999). Matrix metallopro-
teinases (MMPs) are a family of at least 21 zinc-
dependent endopeptidases capable of degrading ECM
components, and other pericellular substrates, for
example, proteinases, protease inhibitors, growth fac-
tors, cytokines, chemokines, and their receptors (Stern-
licht and Werb, 2001). The activity of MMPs is
specifically inhibited by tissue inhibitors of metallopro-
teinases (TIMPs)-1–4, which bind to active MMPs in
1 : 1 molar stoichiometry (Brew et al., 2000). TIMP-1,
TIMP-2, and TIMP-4 are secreted in soluble form,
whereas TIMP-3 is associated with ECM (Pavloff et al.,
1992; Leco et al., 1994; Fariss et al., 1997; Yu et al.,
2000). TIMPs also inhibit the activity of adamalysin
(ADAM) metalloproteinases with a disintegrin and
metalloproteinase domain, and ADAM-TSs with
thrombospondin-like (TS) domains, which play impor-
tant roles in proteolytic processing of protein ectodo-
mains (Amour et al., 1998, 2000; Schlo
¨ndorff and
Blobel, 1999; Kashiwagi et al., 2001). Among the
TIMPs, TIMP-2 and TIMP-3 inhibit all MMPs tested
so far, but with different binding affinities (Brew et al.,
2000). TIMP-3 also inhibits the activity of tumor
necrosis factor-a(TNF-a) converting enzyme (TACE;
ADAM-17), ADAM-10, aggrecanase-1 (ADAM-TS4),
and aggrecanase-2 (ADAM-TS5) (Amour et al., 1998,
2000; Kashiwagi et al., 2001). Accordingly, TIMP-3
inhibits shedding of several cell surface proteins, such as
TNF-a, TNF-RI, syndecan-1 and -4, interleukin-6
receptor, and L-selectin (Smith et al., 1997; Amour
et al., 1998; Hargreaves et al., 1998; Borland et al., 1999;
Fitzgerald et al., 2000). Recent studies have also
identified TIMP-3 as a tumor suppressor, as its
expression is silenced by hypermethylation of the gene
in malignant tumors, such as kidney, lung, colon, breast,
brain, and pancreatic cancers (Bachman et al., 1999;
Loging and Reisman, 1999; Pennie et al., 1999; Ueki
et al., 2000). In addition, TIMP-3 promotes apoptosis in
normal and malignant human cells in culture and in vivo
Received 22 April 2002; revised 2 December 2002; accepted 5 December
2002
*Correspondence: V-M Ka
¨ha
¨ri, Centre for Biotechnology, University
of Turku, Tykisto
¨katu 6B, FIN-20520 Turku, Finland;
E-mail: veli-matti.kahari@utu.fi
Oncogene (2003) 22, 2121–2134
&
2003 Nature Publishing Group
All rights reserved 0950-9232/03 $25.00
www.nature.com/onc
(Smith et al., 1997; Ahonen et al., 1998, 2002; Baker
et al., 1998, 1999). The proapoptotic activity of TIMP-3
has been mapped to N-terminus of the molecule, which
possesses the MMP inhibitory activity (Bond et al.,
2000).
There are six known cell surface receptors with
homologous cytoplasmic sequence called death domain
(Ashkenazi and Dixit, 1999; Wehrli et al., 2000). The
best characterized of these are TNF-RI, FAS (CD95,
APO-1), and TNF-related apoptosis inducing ligand
receptor-1 (TRAIL-RI, DR4). Upon binding of the
respective ligands, death receptors multimerize, which
leads to clustering of their death domains and activation
of downstream signaling (Herr and Debatin, 2001).
Although these receptors share common structural
features, they differ in their mode of activation of
downstream signaling. FAS, TRAIL-RI, and TRAIL-
RII bind an adaptor protein called FAS-associated
death domain (FADD, Mort1) (Yeh et al., 1998), which
recruits and activates procaspase-8 (Nagata and Gold-
stein, 1995; Muzio et al., 1996; Kischkel et al., 2000).
TNF-RI does not bind FADD directly, but requires
engagement of another adaptor protein called TNF-R-
associated death domain (TRADD) before FADD
and procaspase-8 are recruited to the death domain
(Boldin et al., 1996; Hsu et al., 1996). Activation of
caspase-8 in turn results in the activation of effector
caspases, for example, caspase-3 either directly or via
mitochondrial amplification loop leading to cleavage of
death substrates, such as poly (ADP-ribose) polymerase
(PARP), and to apoptosis (Tewari et al., 1995; Cohen,
1997).
In the present study, we show that exposure to
recombinant TIMP-3, N-terminal domain of TIMP-3,
as well as adenoviral expression of TIMP-3 results in the
stabilization of death receptors TNF-RI, FAS, and
TRAIL-RI on the surface of melanoma cells sensitizing
them to apoptosis induced by their ligands. In addition,
we show that TIMP-3 alone induces apoptotic signaling
of these death receptors and that this is mediated by
caspase-8. Stabilization of TNF-RI and FAS was also
noted in vivo in human melanoma xenografts injected
with TIMP-3 adenovirus. Taken together, these results
show that TIMP-3 promotes apoptosis through stabili-
zation and activation of three distinct death receptors in
melanoma cells in culture and in vivo.
Results
Recombinant TIMP-3 and batimastat promote apoptosis
in melanoma cells
We have previously shown, that adenovirally mediated
expression of TIMP-3 promotes apoptosis in melanoma
cells in culture and in vivo and inhibits growth and
angiogenesis in human melanoma xenografts in SCID
mice (Ahonen et al., 1998, 2002). To elucidate the
mechanism of TIMP-3 induced apoptosis, we first
treated human A2058 melanoma cells with recombinant
full-length TIMP-3 and N-terminal domain of TIMP-3
(N-TIMP-3), which harbors the MMP inhibitory and
the apoptosis inducing domain (Bond et al., 2000) under
serum-free conditions for 96 h. Determination of cell
viability by MTT assay showed that full-length TIMP-3
dose-dependently induced cell death in 49 and 93% of
cells with concentrations 25 and 50 nm, respectively
(Figure 1a). Incubation of melanoma cells with N-
TIMP-3 also dose-dependently induced cell death, with
98% of cells killed with concentration 50 nm, whereas N-
terminal MMP inhibitory domain of TIMP-1 (N-TIMP-
1) had no effect on cell viability (Figure 1b). Exposure of
A2058 cells to TIMP-3 and N-TIMP-3 (50 nm) for 72 h
resulted in apoptotic morphological alterations, that is,
shrinkage of the nuclei and condensation of DNA
detected by Hoechst staining, whereas no signs of
apoptosis were detected in cells treated with N-TIMP-
1 (50 nm) or in untreated control cultures (Figure 1d).
Previous observations have shown that synthetic
wide-spectrum MMP inhibitor, batimastat (BB94), also
inhibits growth of melanomas in vivo and shares similar
MMP inhibitory profile with TIMP-3 (Chirivi et al.,
1994; Amour et al., 1998). In this context, we also
examined the effect of BB94 on viability of A2058
melanoma cells. Incubation of A2058 cells with BB94
(6 mm) for 96 h killed 50% of the cells (Figure 1c) and
induced apoptosis in them within 72 h, although not as
potently as TIMP-3 and N-TIMP-3 (Figure 1d).
TIMP-3 stabilizes TNF-RI, FAS, and TRAIL-RI on
melanoma cell surface
To examine the role of death receptors in apoptotic cell
death induced by TIMP-3, human A2058 and SK-Mel-5
melanoma cells were treated with TIMP-3, TIMP-2, and
TIMP-4 under serum-free conditions for 36 h, cell
surface proteins were extracted and subjected to
Western blot analysis for determination of the levels of
death receptors. Treatment with TIMP-3 resulted in
accumulation of TNF-RI, FAS, and TRAIL-RI on the
cell surface, whereas TIMP-2 and TIMP-4 had no effect
on the levels of these death receptors (Figure 2a).
Similarly, exposure of A2058 and WM-266-4 melanoma
cells to N-TIMP-3 increased the levels of TNF-RI, FAS,
and TRAIL-RI on the cell surface, whereas N-TIMP-1
in the same concentration had no effect on the cell
surface levels of the ectodomains of these death
receptors (Figure 2b). In parallel cultures, exposure of
all three melanoma cell lines to BB94 for 36 h resulted in
a dose-dependent increase in the levels of cell surface
TNF-RI, FAS, and TRAIL-RI, although the effect of
BB94 in this respect was less potent than that of TIMP-3
and N-TIMP-3 (Figure 2a, b).
Next, A2058 cells were transduced for 12 h with
recombinant adenoviruses coding for TIMP-3 (RAd-
TIMP-3), TIMP-1 (RAdTIMP-1), TIMP-2 (RAdTIMP-
2), and with adenovirus coding for b-galactosidase
(RAdlacZ) at MOI 20 and 50 and incubated for an
additional 24 h. Dose-dependent accumulation of TNF-
RI, FAS, and TRAIL-RI was noted on the cell surface
of RAdTIMP-3-infected cells, whereas adenoviral ex-
pression of TIMP-1 and TIMP-2 had no effect on the
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levels of cell surface-bound death receptors, as com-
pared to control virus (RAdlacZ)-infected cells
(Figure 2c). Increased expression of FAS and TRAIL-
RI (DR-4) on RAdTIMP-3 infected melanoma cell
surface was also noted by FACS analysis (Figure 2d).
No TRAIL-RII could be detected on A2058 melanoma
cell surface (data not shown). The levels of membrane-
type-1 matrix metalloproteinase (MT1-MMP) on the
cell surface were not markedly altered by any of the
treatments above (Figure 2a–c). The expression of TNF-
RI and FAS mRNAs was not altered by any of the
treatments mentioned above (data not shown).
TIMP-3 sensitizes melanoma cells to death
receptor-mediated apoptosis
Next, we studied whether TIMP-3-induced accumula-
tion of death receptors on surface of melanoma
Figure 1 Induction of apoptosis in melanoma cells by recombinant TIMP-3, N-terminal domain of TIMP-3 and batimastat. (a)
Human A2058 melanoma cells were treated with recombinant TIMP-3 (25 and 50 nm) for 96 h and cell viability was determined with
MTT assay. The mean+s.d. are shown (n¼4). (b) A2058 melanoma cells were treated with N-terminal domain of TIMP-1 (N-TIMP-
1) and N-terminal domain of TIMP-3 (N-TIMP-3) (25 and 50 nm) for 96 h, and cell viability was determined with MTT assay. The
mean+s.d. are shown (n¼4). (c) A2058 cells were treated with batimastat (BB94) (3 and 6 mm) for 96 h and were subjected to MTT
assay. The mean+s.d. are shown (n¼4). Statistical significance was determined by Student’s t-test as compared to untreated cells
(control). (d) A2058 cells were treated with N-TIMP-1, N-TIMP-3, TIMP-3 (50 nm), and BB94 (6 mm) for 72 h and apoptotic cells were
detected with Hoechst staining
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cells affects their susceptibility to apoptosis induced
by death receptor ligands. A2058, SK-Mel-5, and
WM-266-4 melanoma cells were first incubated for
24 h with recombinant TIMP-3 (25 nm) followed by
addition of TNF-a(100 ng/ml), anti-FAS-
antibody (50 ng/ml), and TRAIL (25 ng/ml) to
cultures and determination of cell viability with MTT
assay 24 h later. Incubation of melanoma cells with
TIMP-3 alone for 48 h had no marked effect on
cell viability. Exposure of melanoma cells to TNF-a
alone had no marked effect on the viability of the
cells, whereas the cytotoxic effect of anti-FAS-Ab
and TRAIL was slightly more potent, but did not
reach statistical significance (Figure 3a). However,
treatment with recombinant TIMP-3 significantly
sensitized the melanoma cells to apoptosis induced by
TNF-a, anti-FAS-Ab, and TRAIL (Figure 3a).
Exposure of cells to recombinant N-TIMP-1,
TIMP-2, and TIMP-4 under similar conditions
had no effect on the ability of TNF-a, anti-
FAS-Ab or TRAIL to induce cell death (data not
shown).
To examine, whether adenoviral expression of TIMP-
3 also makes melanoma cells more susceptible to death
receptor-induced apoptosis, A2058 cells were trans-
duced with RAdTIMP-3 and RAdlacZ (MOI 20) for
12 h, followed by a 24 h incubation with TNF-a, anti-
FAS-Ab, and TRAIL in the same concentrations as
used above. Significant sensitization to cell death
induced by these death receptor ligands was detected
in RAdTIMP-3-infected cultures, as compared to
control virus infected cultures (Figure 3b). In accor-
dance with previous observations (Ahonen et al., 1998,
2002), this short postinfection incubation period with
RAdTIMP-3 had limited effect on cell viability
(Figure 3b).
To investigate whether BB94 also sensitizes melanoma
cells to death receptor-mediated apoptosis, A2058 cells
were treated with BB94 (6 mm) for 24 h, followed by the
addition of TNF-a, anti-FAS-Ab, and TRAIL, and the
Figure 2 TIMP-3 stabilizes TNF-RI, FAS, and TRAIL-RI on melanoma cell surface. (a) Human A2058 and SK-Mel-5 melanoma
cells were treated with recombinant TIMP-2, TIMP-3, and TIMP-4 (12.5 and 25 nm), N-terminal domain of TIMP-1 (N-TIMP-1,
25 nm), and batimastat (BB94) (6 mm) for 36 h under serum-free conditions. Cell surface proteins were extracted and subjected to
Western blotting for determination of TNF-RI, FAS, TRAIL-RI, and MT1-MMP levels. (b) Human A2058 and WM-266-4 melanoma
cells were treated with N-TIMP-1, N-TIMP-3 (12.5 and 25 nm), and batimastat (BB94) (3 and 6 mm) for 36 h in serum-free conditions.
Cell surface proteins were extracted and TNF-RI, FAS, TRAIL-RI, and MT1-MMP levels were determined by Western blotting. (c)
A2058 melanoma cells were transduced with adenoviruses coding for b-galactosidase (RAdlacZ), TIMP-1 (RAdTIMP-1), TIMP-2
(RAdTIMP-2), and TIMP-3 (RAdTIMP-3) at MOI 10 and 20 for 12 h, and incubated for 24 h, after which the cell surface proteins
were extracted and the levels of TNF-RI, FAS, TRAIL-RI, and MT1-MMP determined by Western blotting. (d) The levels of FAS and
TRAIL-RI (DR4) on surface of cells infocted with RAdlacZ and RAdTIMP-3, as in (c) were determined by FACS analysis after
immunostaining
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incubations were continued for 24 h. Again, marked
sensitization to death receptor ligand induced cytotoxi-
city was evident in cultures treated with BB94
(Figure 3c).
To confirm induction of melanoma cell apoptosis by
death receptor ligands in the presence of TIMP-3 we
performed terminal dUTP nick end labeling (TUNEL)
and quantitated the number of apoptotic cells with
Figure 3 TIMP-3 sensitizes melanoma cells to apoptosis induced by TNF-a, anti-FAS-Ab and TRAIL. (a) Human melanoma cells
(A2058, SK-Mel-5, and WM-266-4) were pretreated with or without TIMP-3 (50 nm) for 24 h. Subsequently, cells were treated for 24 h
with or without TNF-a(100 ng/ml), anti-FAS-Ab (50 ng/ml), or TRAIL (25 ng/ml) and cell viability was determined with MTT assay.
(b) A2058 melanoma cells were transduced with adenoviruses coding for b-galactosidase (RAdlacZ) and TIMP-3 (RAdTIMP-3) (MOI
20) for 12 h and subsequently incubated for 12 h. Cells were then treated with or without TNF-a(100 ng/ml), anti-FAS-Ab (50 ng/ml),
or TRAIL (25 ng/ml) for 24 h and the number of viable cells was determined with MTT assay. (c) A2058 cells were treated with
batimastat (BB94, 6 mm) for 24 h. Subsequently, cells were treated with TNF-a(100 ng/ml), anti-FAS-Ab (50 ng/ml), and TRAIL
(25 ng/ml) for 24 h and subjected to MTT assay. The mean+s.d. are shown (n¼4). Statistical significance was determined with
Student’s t-test, as indicated, (d) A2058 melanoma cells were treated as above, apoptotic cells were detected by TUNEL labeling and
quantitated with FACS analysis. The percentage of apoptotic cells is shown in parentheses
TIMP-3 induces apoptosis through death receptor
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FACS analysis after similar treatments as above.
Infection of cells with RAdTIMP-3 had slight apoptosis
inducing activity (Figure 3d). Here, slight increase
in the percentage of apoptotic cells was detected in
TNF-atreated cultures and was more evident in cultures
treated with anti-FAS-Ab and TRAIL. However,
the proapoptotic effect of these death receptor
ligands was dramatically increased in RAdTIMP-3-
infected cultures (Figure 3d). Similarly, treatment of
A2058 cells with BB94 for 24 h before addition of TNF-
a, anti-Fas-Ab, and TRAIL markedly potentiated the
proapoptotic effect of these death receptor ligands
(Figure 3d).
TIMP-3-induced apoptosis is mediated by caspase-8
To examine whether accumulation of cell surface death
receptors as a result of exposure to TIMP-3 results in
activation of their downstream signaling, we treated
A2058, SK-Mel-5, and WM-266-4 melanoma cells with
recombinant TIMP-3 (50 nm) for 96 h and determined
the activation of the most proximal caspase, caspase-8, a
key mediator in death receptor-mediated apoptotic
signaling (Cohen, 1997). Caspase-8 activation, detected
as reduction in the levels of proform of caspase-8 was
detected in TIMP-3-treated cultures, indicating activa-
tion of death receptors (Figure 4a). In addition,
increased cleavage of death substrate PARP was noted
in all TIMP-3-treated melanoma cell cultures, as a
marker of activation of caspase-3, the downstream
target of caspase-8 (Figure 4a). Next, we transduced
A2058 melanoma cells with RAdlacZ, RAdTIMP-1,
and RAdTIMP-3 (MOI 10 and 20) for 12 h and
incubated cells for an additional 72 h. Reduction in
procaspase-8 levels was detected in RAdTIMP-3 in-
fected cultures associated with increased cleavage of
PARP, whereas infection of cells with RAdlacZ and
RAdTIMP-1 had no effect on procaspase-8 or PARP
Figure 3 Continued
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cleavage (Figure 4a). The levels of constitutively
expressed heat shock cognate protein 70 (Hsc70)
remained unaltered with all treatments.
Next, we studied further the role of caspase-8 in
TIMP-3-induced apoptosis by utilizing specific caspase-
8 inhibitor (Z-IETD-FMK). Cells were treated with N-
TIMP-3 (25 nm) for 24 h followed by addition of caspase
inhibitor (20 mm), and cell viability was assessed 72 h
later. Induction of cell death by N-TIMP-3 was potently
inhibited by caspase-8 inhibitor, as no significant
difference in cell viability was detected between un-
treated cultures and cultures treated with the combina-
tion of N-TIMP-3 and caspase-8 inhibitor (Figure 4b).
We also studied the role of caspases in apoptosis
induced by adenovirally delivered TIMP-3. A2058
cells were transduced with RAdTIMP-3 and RAdlacZ
(MOI 20), followed by addition of Z-IETD-FMK,
and pan-caspase inhibitor (Z-DEVD-FMK), which
inhibits the activity of caspases-3, -6, -7, and -8, 24 h
later. Assay of cell viability 96 h after the infection
indicated that cell death in RAdTIMP-3-infected
cultures was inhibited by 59% by caspase-8 inhibitor
and by 51% by pan-caspase inhibitor (Figure 4c).
Neither caspase inhibitor had effect on viability of
cells infected with control adenovirus RAdlacZ
(Figure 4c).
Figure 4 TIMP-3 induces apoptosis through activation of caspase-8. (a) Human melanoma cells (A2058, SK-Mel-5, and WM-266-4)
were treated for 96h with recombinant TIMP-3 (50 nm) or transduced with adenoviruses harboring genes for b-galactosidase
(RAdlacZ), TIMP-1 (RAdTIMP-1), and TIMP-3 (RAdTIMP-3) (MOI 10 and 20). Subsequently, whole-cell lysates were extracted and
equal amounts of proteins were subjected to Western blot analysis with anti-caspase-8 antibody, anti-PARP antibody, and anti-heat
shock cognate 70 (Hsc70) antibody. The level of uncleaved caspase-8 is shown relative to levels in untreated control cells (left panels)
and RAdLacZ (MOI 10) infected cultures (right panel). The percentage of cleaved form of PARP out of total PARP is shown. (b)
Human A2058, SK-Mel-5, and WM-266-4 melanoma cells were treated with N-TIMP-3 (25nm) for 24 h. Subsequently, caspase-8
inhibitor (Z-IETD-FMK) (20 mm) was added, incubations were continued for an additional 72 h, and cell viability was assessed with
MTT assay. (c) A2058 melanoma cells were transduced with RAdTIMP-3 and RAdlacZ (MOI 20) for 12 h and incubated for another
12 h. Subsequently, caspase-8 inhibitor (Z-IETD-FMK) and pan-caspase inhibitor (Z-DEVD-FMK) (20 mmeach) were added and the
number of viable cells was quantitated with MTT assay 96 h post-transduction. The mean+s.d. are shown (n¼4). Statistical
significance was determined by Student’s t-test, as indicated. (d) A2058 cells were transduced with RAdTIMP-3 and RAdlacZ (MOI
20) for 12 h and incubated for another 12 h. Subsequently, caspase-8 inhibitor (Z-IETD-FMK) and pan-caspase inhibitor (Z-DEVD-
FMK) (20 mmeach) were added. At 72 h after infections, cells were stained for cleaved caspase-3 and counterstained with Hoechst for
detection of apoptotic nuclear morphology
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To further examine the role of caspase-8 in TIMP-3-
induced apoptosis, we infected A2058 melanoma cells
with RAdTIMP-3 and RAdlacZ as above, added
caspase-3 and pan-caspase inhibitors 24 h postinfection,
and immunostained cells for cleaved caspase-3 and
counterstained the same cultures with Hoechst 48 h
later, that is, 72 h after adenoviral infections. Cells
positive for activated caspase-3 and with apoptotic
nuclear morphology were detected in RAdTIMP-3-
infected cultures (Figure 4d). In contrast, treatment
with caspase-8 inhibitor (Z-IETD-FMK) and pan-
caspase inhibitor (Z-DEVD-FMK) entirely inhibited
activation of caspase-3 and apoptosis in RAdTIMP-3
infected A2058 cultures (Figure 4d).
Adenoviral TIMP-3 expression results in TNF-RI
stabilization in melanoma cells in vivo
We have recently observed that adenoviral delivery of
TIMP-3 induces apoptosis in melanoma cells in vivo and
inhibits growth of human melanoma xenografts in
SCID mice (Ahonen et al., 2002). In this context, we
examined, whether TIMP-3 also stabilizes death recep-
tors in vivo. A2058 melanoma cells were injected into
subcutaneous space in SCID/SCID mice), tumors were
allowed to reach the size of 50–100 mm
3
, and were then
injected with RAdTIMP-3 and the empty control virus
RA66 (1.4 10
9
PFU each in 100 ml PBS; n¼4 for each
group) every 24 h for 3 consecutive days. At 24 h after
the last adenovirus injection, tumors were examined for
the expression of TIMP-3, TNF-RI, FAS, and for the
presence of active caspase-3. Significant staining for
TNF-RI and FAS was detected in RAdTIMP-3-injected
tumors, adjacent to areas containing TIMP-3 expressing
cells in parallel sections (Figure 5, right panels). No
TIMP-3, TNF-RI, or FAS immunostaining was de-
tected in tumors injected with empty control virus
(RAd66) (Figure 5, left panels). Apoptotic cells with
nuclear condensation and fragmentation were detected
adjacent to regions with cells expressing TIMP-3, TNF-
Figure 4 Continued
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RI, and FAS (Figure 5, right panels, arrows). Staining
for cleaved, active form of caspase-3 was seen in
apoptotic cells in the proximity of TIMP-3-positive cells
showing TNF-RI and FAS expression on cell surface
providing evidence that accumulation of TNF-RI and
FAS is an early event during TIMP-3-induced apoptosis
and is lost when cells undergo apoptosis.
Discussion
The present study was conducted to elucidate the role of
cell surface death receptors in TIMP-3-induced apopto-
sis in human melanoma cells. Our results show, that
recombinant TIMP-3, as well as N-terminal domain of
TIMP-3 at same molar concentration (25 and 50 nm)
Figure 5 Adenovirally delivered TIMP-3 stabilizes TNF-RI and FAS in human melanoma cells in vivo. A2058 melanoma cells
(1 10
6
) were injected subcutaneously in the back of SCID/SCID mice and allowed to reach the size of 50–100 mm
3
. Subsequently,
tumors were injected with recombinant adenovirus for TIMP-3 (RAdTIMP-3) or with empty control adenovirus (RAd66) (1.410
9
PFU each) every 24 h for 3 consecutive days (n¼4 for each group). Tumors were harvested 24 h after the last injection and parallel
tumor sections were analysed for TIMP-3, TNF-RI, FAS, and cleaved caspase-3 by immunostaining with specific antibodies. Scale bar:
50 mm. Arrows indicate the same apoptotic area in parallel sections (right panels) and arrowheads indicate same blood vessel in
adjacent sections (left panels)
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induce apoptosis in melanoma cells. We also show, that
exposure of three metastatic melanoma cell lines to
exogenous recombinant full-length TIMP-3 and N-
terminal domain of TIMP-3, as well as to adenovirally
expressed endogenous TIMP-3 results in stabilization of
three distinct death receptors, TNF-RI, FAS, and
TRAIL-RI on cell surface and makes these cells more
susceptible to apoptosis induced by their respective
ligands. Our results also show, that TIMP-3-induced
accumulation of death receptors results in activation of
their apoptotic signaling, as detected by activation of
caspase-8, caspase-3, and cleavage of downstream death
substrate PARP. In addition, our results show, that the
apoptotic cell death induced by TIMP-3 is dependent on
the activity of caspase-8. Furthermore, we show, that
adenoviral expression of TIMP-3 in human melanoma
xenografts in vivo results in stabilization of TNF-RI and
FAS, and in activation of caspase-3 in melanoma cells.
Taken together, these results provide evidence that
turnover of cell surface death receptors in the absence of
TIMP-3 is rapid, minimizing ligand binding, death
receptor multimerization, and activation. However, as a
result of TIMP-3-induced stabilization and increased
availability of death receptors, binding of death ligands,
multimerization and activation of receptors takes place
(Figure 6). Our results showing that TIMP-3 alone can
induce activation of death receptor signaling, also
suggest that accumulation of death receptors on cell
surface results in multimerization and activation of
death receptors in the presence of limited amount of
their ligands. This is supported by our observation, that
soluble TNF-aand FasL cannot be detected in
conditioned medium of melanoma cells (data not
shown). Previous studies also suggest, that death
receptor oligomerization and activation may take place
even in the absence of death ligands, when the cell
surface becomes saturated with death receptors leading
to (auto) multimerization and subsequent activation of
apoptotic signaling (Boldin et al., 1995; Chan et al.,
2000).
Recent studies have shown that TIMP-3 induces
apoptosis in several cell types (Ahonen et al., 1998, 2002;
Baker et al., 1998, 1999). Our previous observations also
showed that adenoviral expression of TIMP-3 inhibits
adhesion of melanoma cells to ECM prior to induction
of apoptosis (Ahonen et al., 1998). As TIMP-3 binds to
ECM, specifically to sulfated glycosaminoglycans via its
N- and C-terminal domains (Langton et al., 1998; Yu
et al., 2000), it is possible, that it also promotes
apoptosis by interfering with survival signal provided
by ECM to cells. However, the proapoptotic activity of
TIMP-3 has been mapped to three loops in the N-
terminus of the molecule necessary for the inhibition of
metalloproteinase activity, suggesting that TIMP-3
induces apoptosis by inhibiting proteolytical processing
of ECM components or cell surface proteins (Bond et al.,
2000). In addition, our results here showed that N-
TIMP-3 and TIMP-3 are equally potent in inducing
apoptosis in melanoma cells, indicating that matrix
binding at least through C-terminal domain is not
necessary for the proapoptotic effect of TIMP-3. This
notion is also supported by our recent observations
Figure 6 Schematic illustration of suggested mechanism of TIMP-3-induced apoptosis. In the absence of TIMP-3, metalloproteinase-
dependent shedding of death receptors from cell surface results in suppression of death receptor signaling and promotes cell survival
(left panel). In the presence of TIMP-3, death receptor shedding is inhibited resulting in ligand binding, oligomerization and activation
of death receptors, and in subsequent activation of apoptotic signaling pathway and cell death (right panel)
TIMP-3 induces apoptosis through death receptor
M Ahonen
et al
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showing that adenoviral expression of TIMP-3 by
melanoma cells results in apoptosis of uninfected by-
stander cells and that this effect is mediated by soluble
TIMP-3 in conditioned medium of RAdTIMP-3-in-
fected cells (Ahonen et al., 2002).
It has been reported that expression of TIMP-3
stabilized TNF-RI on surface of stably transfected colon
carcinoma cells, which became more susceptible to
apoptosis by serum deprivation, although no evidence
for activation of TNF-RI was provided (Smith et al.,
1997). In our study, TIMP-1, TIMP-2, and TIMP-4 had
no effect on death receptor levels or cell viability,
suggesting that shedding of death receptors on melano-
ma cells involves activity of proteinases other than
MMPs. In addition, the activity of the members of
Adamalysin gene family, at least ADAM-10 (human
homologue of Kuzbanian) is inhibited by TIMP-1
(Amour et al., 2000). Shedding of TNF-RI and TNF-
RII can be inhibited by blocking the expression of
TACE (ADAM-17) (Solomon et al., 1999), and the
activity of TACE is inhibited by batimastat (BB94) and
TIMP-3, but not by other TIMPs (Amour et al., 1998).
However, there is no direct evidence at present that
TACE can shed also FAS or TRAIL-RI. It is therefore
possible that the proapoptotic effect of TIMP-3 is
mediated by inhibition of several sheddases involved in
turnover of death receptors. Our data showing stabiliza-
tion of death receptors and induction of apoptosis also
by MMP inhibitor batimastat, provide strong evidence
that MMP-dependent sheddase activity plays an im-
portant role in turnover of death receptors on cell
surface and that inhibition of this event by small
molecule inhibitor or TIMP-3 sensitizes cells to death
receptor-mediated apoptosis.
Recently, activation of caspase-8 by TIMP-3 was
reported, and TIMP-3 induced apoptosis was inhibited
by dominant-negative FADD and crmA, which block
caspase-8 activation and activity, respectively (Bond
et al., 2002). These data clearly point out that TIMP-3
induces apoptosis upstream of FADD, which binds
death receptors directly or through an adaptor molecule
and is essential for death receptor-induced apoptosis.
Furthermore, blocking death receptor ligand binding by
TNF-a-neutralizing antibody, Fas/Fc, or soluble
TRAIL receptor had no effect on TIMP-3-induced
apoptosis (Bond et al., 2002). Together with our data,
showing that TIMP-3 promotes stabilization of three
distinct death receptors, these observations suggest that
inhibition of ligand binding and activation of one death
receptor alone may not be sufficient to inhibit TIMP-3-
induced apoptosis if ligand(s) of other receptor(s) are
present.
Recently, enhancement of epithelial cell apoptosis
because of unscheduled ECM degradation associated
with early activation of gelatinase-A (MMP-2) during
mammary gland involution in TIMP-3-deficient mice
was described, and this could be reversed by adminis-
tration of recombinant TIMP-3 and chemical MMP
inhibitor ilomastat, which has inhibitory activity against
both MMPs and ADAMs (Fata et al., 2001). However,
our previous observations, and the results of the present
study show that both adenovirally mediated overexpres-
sion of TIMP-3 and exposure to recombinant TIMP-3
promote apoptosis in various type of cells in culture and
in vivo (Ahonen et al., 1998, 2002; Baker et al., 1998;
Bond et al., 2000, 2002). Nevertheless, together these
observations show that TIMP-3 may affect cell survival
in a different manner depending on the concentration
and the cellular environment.
Increased serum levels of FasL in melanoma patients
have been shown to correlate with poor prognosis and
metastatic disease (Soubrane et al., 2000; Ugurol et al.,
2001). In addition, higher expression of FasL is found in
melanoma metastases than in primary melanomas
(Ekmekcioglu et al., 1999; Terheyden et al., 1999).
Melanoma metastases display decreased FAS expression
as compared to primary melanomas and downregula-
tion of FAS may favor metastatic behavior of melano-
ma cells (Owen-Schaub et al., 1998; Soubrane et al.,
2000). Accumulation of FAS on cell surface by TIMP-3
may have functional significance in terms of therapy,
since stabilization of FAS by TIMP-3 is likely to render
these cells susceptible to apoptosis by FasL produced by
tumor cells themselves or other cells. Recently, cell
surface cleavage of FasL in tumor cells by metallopro-
teinase activity has been documented (Mitsiades et al.,
2001). Inhibition of this process by TIMP-3 may also
have therapeutic significance as FasL secretion by tumor
cells has been suggested to play a major role in immune
escape by FAS-positive tumor infiltrating immune
effector cells (O’Connell et al., 1999).
We have recently shown that adenoviral delivery of
TIMP-3 to human melanoma tumors established in
SCID mice inhibits their growth and induces apoptosis
in these cells in vivo (Ahonen et al., 2002). The
observations presented here show that adenoviral
expression of TIMP-3 in vivo in human melanoma
xenografts results in stabilization of TNF-RI and FAS,
activation of caspase-3, and apoptosis in tumor cells
adjacent to cells expressing TIMP-3 providing evidence
that inhibition of death receptor shedding by TIMP-3
also promotes apoptosis of malignant cells in vivo.Itis
conceivable, that tumor-targeted gene delivery of TIMP-
3 in combination with administration of death receptor
ligand(s) may provide a novel approach to cancer gene
therapy. Inactivation of specific death receptor signaling
in malignant cells appears one way of acquiring
resistance to apoptosis (Owen-Schaub et al., 1998;
Soubrane et al., 2000). In this respect, the ability of
TIMP-3 to stabilize and activate multiple death recep-
tors may provide a novel way of inducing death of
cancer cells via activation of apoptotic signaling through
any one of the three death receptors.
Materials and methods
Melanoma cell cultures
Melanoma cell lines A2058, SK-Mel-5, and WM-266-4,
established from the metastases of human malignant melano-
ma, were obtained from the American Type Culture Collection
(Manassas, VA, USA) and cultured in Dulbecco’s modified
TIMP-3 induces apoptosis through death receptor
M Ahonen
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Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum (FCS), 2 mmglutamine, 100 IU/ml penicillin-G and
100 mg/ml streptomycin.
Adenoviral cell infections
Construction and characterization of replication-deficient
adenoviruses containing the coding region of human TIMP-1
(RAdTIMP-1), TIMP-2 (RAdTIMP-2), or TIMP-3 (RAd-
TIMP-3) genes driven by CMV IE promoter has been
described previously (Baker et al., 1996, 1998). Recombinant
replication-deficient adenovirus RAdLacZ (RAd35), which
contains the Escherichia coli b-galactosidase (LacZ) gene under
the control of cytomegalovirus immediate-early (CMV IE)
promoter, and corresponding empty adenovirus (RAd66)
were kindly provided by Dr Gavin WG Wilkinson (University
of Cardiff, Wales) (Wilkinson and Akrigg, 1992). Virus
propagation and titer determination of recombinant
adenoviruses were performed as described previously (Ahonen
et al., 1998). Melanoma cells in culture were infected
at different MOI by incubating cells with adenoviruses for
16 h, as described previously (Ahonen et al., 1998). The cell
cultures were then washed twice with PBS and fresh media
were added.
Recombinant proteins and caspase inhibitors
Recombinant N-TIMP-3, N-TIMP-1, TIMP-2, and TIMP-4
were expressed in E.coli and refolded from the inclusion
bodies, as described previously (Kashiwagi et al., 2001).
Recombinant TIMP-3 was purchased from R&D Systems
(Minneapolis, MA, USA). TNF-awas obtained from Sigma
(St Louis, MO, USA), FAS activating antibody (CH-11) was
purchased from MBL International Corporation (Watertown,
MA, USA), and recombinant soluble FLAG-tagged TRAIL
was from Alexis corporation (La
¨uflingen, Switzerland).
TRAIL was incubated for 15 min on ice with crosslinking
anti-FLAG antibody M2 (Sigma, St Louis, MO, USA) prior
to adding to cell culture medium. Batimastat (BB94) was
kindly provided by British Biotech Inc. (Oxford, UK). Z-
DEVD-FMK (caspase-3 inhibitor II) and Z-IETD-FMK
(caspase-8 inhibitor II) were purchased from Calbiochem
(Darmstadt, Germany).
Western blot analysis of cell surface proteins
For analysis of death receptor levels, cell surface proteins were
extracted, as described previously (Mitsiades et al., 1999).
Briefly, cells were maintained on cell culture dishes, washed
with PBS, and incubated at room temperature with 0.5 ml of
0.5 mg/ml PBS of EZ-Linkt-Sulfo-NHS-LC-Biotin (Pierce,
Rockford, IL, USA) for biotinylation of the ectodomains of
cell surface proteins. Cells were then washed with PBS and
collected in lysis buffer containing 50 mmTris-HCl (pH ¼8),
120 mmNaCl and 1% Ipecal supplemented with Completet
proteinase inhibitor mixture (Boehringer-Mannheim). Subse-
quently, supernatants were collected after centrifugation and
equal amounts of extracts were used to precipitate the
biotinylated proteins with Immunopure
s
immobilized strepta-
vidin. After centrifugation and washes with PBS cell surface
proteins were released by boiling in Laemmli buffer containing
5% b-mercaptoethanol. Aliquots of membrane proteins were
fractionated electrophoretically on SDS–polyacrylamide
(10%) gels, transferred to nitrocellulose filter (Amersham,
England) and incubated at 201C with polyclonal antibodies
against FAS (C-20), TRAIL-RI (DR4) (H-130)(Santa Cruz
Biotechnology, Santa Cruz, CA, USA), and MT1-MMP
(Lehti et al., 1998) or with monoclonal antibody against
TNF-RI (H-5) (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) for 12 h at dilution of 1 : 50 to 1 : 1000. The filters
were then incubated with secondary anti-rabbit or anti-mouse
horseradish peroxidase-conjugated antibodies for 1 h at
room temperature, subjected to ECL reaction (Amersham
Corp., UK), and positive labeling was detected with auto-
radiography.
Surface expression analysis of FAS and TRAIL-RI
Melanoma cells (0.5 10
6
) were infected with RAdTIMP-3
and RAdlacZ for 12 h and incubated for additional 24 h. Cells
were then harvested with 4 mmEDTA, washed with PBS, and
blocked with 1% BSA in PBS for 30 min. Cells were then
incubated with specific antibodies against Fas (MBL) or
TRAIL-RI (DR4, Alexis) in 1% BSA in PBS for 30 min
followed by washing with PBS. Finally, cells were incubated
with Alexa 488-conjugated goat anti-mouse IgG (Molecular
Probes) for 30 min. After washes, cells were analysed on a
FACScan flow cytometer.
Analysis of cell viability and apoptosis
For determination of cell viability, 1 10
4
cells were seeded on
96-well plates and incubated for different periods of time after
the adenoviral infection or addition of different recombinant
proteins. The number of viable cells was determined by
CellTiter 96tAQueous nonradioactive cell proliferation assay
(Promega, Madison, WI, USA). For analysis of nuclear
morphology, cells were cultivated on coverslips, fixed with
4% paraformaldehyde, stained with Hoechst 33342 (10 mg/ml)
and analyzed for nuclear morphology with fluorescent micro-
scopy. For detection of activated caspase-3, cultured cells were
fixed by acetone and stained with cleaved caspase-3-specific
antibody (1 : 300) (Cell Signaling Technology Inc., Beverly,
MA, USA), followed by visualization with anti-rabbit–FITC
labeled secondary antibody. DNA fragmentation was detected
by staining cells with Apoptosis Detection System, Fluorescein
(Promega, Madison, WI, USA) and quantitated by FACS
analysis.
Detection of caspase-8 activation and cleavage of PARP
For the determination of caspase-8 and PARP cleavage, 20 mg
of whole-cell lysates was fractionated on 15 and 10% SDS–
polyacrylamide gels and analyzed by Western blotting using
polyclonal antibodies against PARP (Sigma), caspase-8 (C15
caspase-8 antibody, a kind gift from Peter Krammer, German
Cancer Research Center, Heidelberg, Germany), and Hsc70
(StressGen). Autoradiograms were quantitated with Micro-
computer Imaging Device version M4 (Imaging Research
Inc.), and the resulting measurements were corrected for Hsc70
protein levels.
Adenoviral infection and analysis of human melanoma
xenografts
All experiments with mice were performed according to
institutional animal care guidelines and with permission of
the animal test review board of the University of Turku,
Finland. For in vivo experiments, tumors were established by
injecting 1 10
6
A2058 cells subcutaneously to the back of
SCID/SCID mice and allowing tumors to grow for 14 days till
they reach the size of 50–100 mm
3
(Ahonen et al., 2002).
Tumors were then injected with adenoviruses (1.4 10
9
PFU)
in 100 ml PBS every 24 h for 3 days (n¼4 for each adenovirus),
24 h after last injection tumors were harvested, fixed in
TIMP-3 induces apoptosis through death receptor
M Ahonen
et al
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Oncogene
formaldehyde and embedded in paraffin. For immunohisto-
chemical staining of TIMP-3, TNF-RI, FAS, and cleaved
caspase-3, tumor sections were pretreated with boiling for
10 min 0.1 mcitric saline and stained with 1 : 50 dilution of
mouse monoclonal antibody identifying human TIMP-3 (Ab-
1)(Oncogene Research Products, San Diego, CA, USA), 1 : 50
dilution of anti-human TNF-RI antibody (H-5) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), 1 : 100 dilution of
rabbit polyclonal anti-human FAS antibody (Santa Cruz
Biotechnology), and 1 : 300 dilution of antibody specific for
cleaved caspase-3 (Cell Signaling Technology, Beverly, MA,
USA). Primary antibodies were visualized with Strept
ABComplex/HRP, Duet, Mouse/Rabbit (DAKO, Glostrup,
Denmark), secondary antimouse antibody was neutralized
with 15 min incubation with mouse serum. Staining with anti-
TIMP-3 monoclonal antibody was detected with Histomouset
SP (AEC) (Zymed, San Francisco, CA, USA) allowing the use
of mouse-monoclonal antibodies in immunostaining of mouse
tissues.
Acknowledgements
We thank Ms Hanna Haavisto, Ms Marjo Hakkarainen, and
Ms Sari Pitka
¨nen for their skillful technical assistance. This
study was supported by grants from the Academy of Finland,
Sigrid Juse
´lius Foundation, the Cancer Foundation of Fin-
land, and Turku University Central Hospital, and by research
contract with Finnish Life and Pension Insurance Companies,
and the Wellcome Trust Grant 057508 and NIH Grant
AR40994 to Hideaki Nagase. Matti Ahonen and Minna
Poukkula are students in Turku Graduate School of Biome-
dical Sciences.
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