The Activation of the c-Jun N-terminal Kinase and p38
Mitogen-activated Protein Kinase Signaling Pathways Protects HeLa
Cells from Apoptosis Following Photodynamic Therapy with
Hypericin*
Zerihun
Assefa
,
Annelies
Vantieghem
§¶,
Wim
Declercq
,
Peter
Vandenabeele
**,
Jackie R.
Vandenheede

,
Wilfried
Merlevede
,
Peter
de Witte§, and
Patrizia
Agostinis
§§
From the
Division of Biochemistry, Faculty of
Medicine, KULeuven, Herestraat 49, § Laboratory for
Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmacy,
KULeuven, Van Evenstraat 4, B-3000 Leuven, Belgium and the
Department of Molecular Biology, Flanders Interuniversity
Institute for Biotechnology (VIB), University of Gent (UG),
Ledeganckstraat 35, B-9000 Gent, Belgium
 |
ABSTRACT |
In this study, we elucidate signaling pathways
induced by photodynamic therapy (PDT) with hypericin. We show that PDT
rapidly activates JNK1 while irreversibly inhibiting ERK2 in several
cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
(zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone
(zDEVD-fmk) protect cells against apoptosis and inhibit
DEVD-specific caspase activity and PARP cleavage without affecting JNK1
and p38 MAPK activations. Conversely, stable overexpression of CrmA,
the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on
PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell
transfection with the dominant negative inhibitors of the c-Jun
N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment
with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A
similar increase in PDT-induced apoptosis was observed by expression of
the dual specificity phosphatase MKP-1. The simultaneous inhibition of
both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more
pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor
PD169316 causes faster kinetics of DEVD-caspase activation and PARP
cleavage and strongly oversensitizes the cells to apoptosis
following PDT. These observations indicate that the JNK1 and p38 MAPK
pathways play an important role in cellular resistance against
PDT-induced apoptosis with hypericin.
 |
INTRODUCTION |
Mitogen-activated protein kinases
(MAPKs)1 are proline-directed
Ser/Thr protein kinases activated by dual phosphorylation on both a
tyrosine and a threonine residue (1). These enzymes are critical
components of a complex intracellular signaling network that ultimately
regulates gene expression in response to a variety of extracellular
stimuli. The three well known mammalian MAPK families are: the
extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal
kinases/stress-activated protein kinases (JNKs/SAPKs), and the p38
MAPK. Each of these enzymes is a target for discrete but closely
related phosphorylation cascades in which the sequential activation of
three kinases constitutes a common signaling module (2). The best
characterized MAPK pathway is the Ras/Raf/MEK cascade leading to the
activation of ERK1/2 in response to growth factors (3). JNK and p38
MAPK are key mediators of stress signals and inflammatory responses
evoked by a variety of agents such as UV- and
-irradiation, heat
shock, osmotic stress, and inflammatory cytokines (4, 5). JNKs are
activated by the dual specificity kinases, MKK4/SEK1 (6) and MKK7 (7), while p38 MAPKs are activated by the MKK3/6 homologues (8). Once
activated, JNKs mediate the phosphorylation and activation of the
transcription factors c-Jun, ATF2, and Elk1 (9, 10). The p38 MAPK
cascade is likewise involved in the transcriptional regulation of ATF2
and Elk1 (11) as well as in the activation of MAPKAP2/3 kinases, which
in turn phosphorylate small heat shock proteins (12, 13).
Because of the strong activation of JNK and p38 MAPK observed in cells
treated with several stress signals that ultimately lead to apoptosis,
considerable attention has recently been focused on the potential role
of these kinases in apoptotic signaling. A causative link between the
JNK/p38 MAPK signaling pathways and apoptosis has been suggested by
several studies. In some reports, it was shown that overexpression of
constitutively active forms of MEKKs (14), the upstream regulators of
MKK4/SEK1, or MKK6 (15) results in apoptosis. Xia et al.
(16) reported that in PC12 cells, JNK and p38 MAPK play a critical role
in mediating apoptosis caused by nerve growth factor withdrawal.
The JNK pathway also seems to be required for the induction of
apoptosis by ceramide,
- and UVC-irradiation, some chemotherapeutic
drugs (17-19), as well as for the Daxx-mediated, Fas-induced cell
death (20). Inversely, other reports have suggested that the activation
of JNK is not mechanistically implicated in the apoptotic process. Inhibition of the JNK pathway by the expression of the dominant negative forms of MEKK1, SEK1, or c-Jun mutant did not prevent Fas- or
TNF-mediated cell death (21, 22). Similarly, a recent study has shown
that the activation of JNK/p38 MAPK does not correlate with apoptosis
induced by the detachment of epithelial cells (23). Furthermore,
thymocytes from sek1
/
mice were found to be
more susceptible to apoptosis induced by Fas and CD3 than their wild
type counterparts (24), suggesting that the JNK pathway may also have a
protective function. In conclusion, the exact role of JNK and/or p38
MAPK in programmed cell death remains highly ambiguous. Moreover, these
reports stress the dependence of specific cellular responses (death or
survival) on the cellular background as well as on the causative agent.
Photodynamic therapy (PDT) is a new cancer treatment based on the topic
or systemic application of a photosensitizing agent that accumulates in
hyperproliferating benign and malignant tissues (reviewed in Ref. 25).
Upon irradiation with tissue-penetrating light, the photosensitizer is
activated and generates reactive oxygen species, which then cause cell
death. PDT is a promising alternative to conventional cancer treatments
involving cytotoxic drugs or ionizing radiation, as it combines a
minimal systemic toxicity with a highly selective photodynamic
destruction of tumor cells. However, the molecular mechanism underlying
its antitumor activity is poorly understood. PDT with porphyrin and a
porphyrin derivative has recently been reported to induce both c-Jun
and c-Fos expression (26) and activation of the JNK/p38 MAPK signaling pathways (27, 28), but a causative link between these observations and
the PDT-induced cytotoxicity is still missing.
In the present study, we have used the photodynamic agent hypericin as
a stress stimulus and investigated its potential signaling to the
different MAPK cascades. Hypericin is a photoactive natural pigment
extracted from plants of the genus Hypericum with a
phenanthroperylenequinone structure. Interest in this compound was
renewed in recent years because of its potential as a photosensitizing
anticancer drug. We (29-31) and others (32, 33) have shown recently
that photo-activated hypericin has a powerful in vivo
antitumor activity. Although hypericin has also been reported to cause
apoptosis in various cancer cell lines (34, 35), the signal
transduction pathways involved have never been investigated.
Here we show that PDT with hypericin leads to a strong and sustained
activation of both JNK1 and p38 MAPKs, while causing a drastic and
irreversible inhibition of ERK2 in all cancer cell lines tested.
Specifically in HeLa cells, we show that hypericin-induced apoptosis
requires a DEVD-directed caspase activity, while the activation of the
JNK/p38 MAPK pathways occurs via a caspase-independent mechanism and is
not related to the process of apoptosis. Furthermore, by expression of
dominant negative mutants of components of these stress-induced kinase
signaling pathways, or by the use of the p38 MAPK inhibitor PD169316,
we show that concomitant inhibition of the JNK and p38 MAPK
significantly enhances cell death. These observations indicate that the
JNK/p38 MAPK pathways contribute to a survival response that
counteracts PDT-induced apoptosis in HeLa cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
GST-c-Jun1-223 and the polyclonal antibodies to
the human JNK1 and ERK2 were prepared as described before (36).
Antiphospho-p38 MAPK (Thr180/Tyr182) monoclonal
antibody, which specifically recognizes the phosphorylated form of the
kinase, was purchased from New England Biolabs, Inc. (Beverly, MA).
Protein A-TSK was from Affiland (Liege, Belgium). Myelin basic protein
and Hoechst 33342 were purchased from Sigma, EGF from Boehringer
Mannheim (Mannheim, Germany), and [
-32P]ATP from
Amersham Pharmacia Biotech (Uppsala, Sweden). Mouse anti-human PARP
antibody was purchased from Biomol (Plymouth, PA). Caspase-3 antibody
was from Santa Cruz (Santa Cruz, CA). zVAD-fmk and zDEVD-fmk were
purchased from Enzyme Systems Products (Livermore, CA). DEVD-amc was
purchased from the Peptide Institute, Inc. (Osaka, Japan). The p38 MAPK
inhibitor PD169316 was purchased from Calbiochem (Bierges, Belgium).
Cell Culture and PDT Treatment--
All cell lines used in these
study were maintained in Dulbecco's modified Eagle's medium
supplemented with 2 mM L-glutamine, 1%
penicillin/streptomycin solution, and 10% FCS (Life Technologies Inc.,
Paisley, Scotland). Cells were incubated at 37 °C in 5% CO2. Preparation and storage of hypericin and cell
photosensitization with hypericin were performed as described elsewhere
(29). Briefly, cells were preincubated for 16 h with different
concentrations of hypericin in culture medium containing 10% FCS in
strictly subdued light conditions (<1 microwatt/cm2). Then
cells were irradiated in hypericin-free medium for 15 min by placing
the plates on a plastic diffuser sheet 5 cm above a set of seven
L18W/30 fluorescent lamps (Osram, Berlin, Germany). At the surface of
the diffuser, the uniform fluence rate was 4.5 milliwatts/cm2 (corresponding to a light dose of 4 J/cm2). In the experiments where EGF was used, the cells
were first starved for 48 h in serum-free medium. Preparation of
cell extracts at the end of incubation periods was carried out exactly
as described before (36). Protein concentrations were estimated with
BCA (Pierce).
Protein Kinase Assays--
JNK1 and ERK2 activities were
measured by immunocomplex kinase assays as described in Ref. 36, with
the exception that the kinase buffer used here was 20 mM
MOPS, pH 7.4, 15 mM MgCl2, 2 mM
EGTA, 1 mM 1 dithiothreitol, 0.1% Triton X-100, 1 mM Na3VO4. The p38 MAPK activation
was determined by Western blot analysis using antiphospho-p38 MAPK antibody.
Evaluation of Apoptosis and Caspase Activity--
Following
treatment as described above, cells were incubated in the dark for
24 h, and apoptosis was evaluated by fluorescent microscopic
analysis of fragmented nuclei stained with Hoechst 33342. Caspase
activity assays were performed as described (37).
Western Blot Analysis--
Samples (20-40 µg of protein) from
cell lysates were separated by SDS-polyacrylamide gel electrophoresis
and electrophoretically transferred to nitrocellulose or polyvinylidene
difluoride membranes (Bio-Rad). The membranes were blocked in Blotto
(5% non-fat dry milk in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20)) for 1 h at room temperature
and incubated with primary antibody for 2 h at room temperature
(anti-PARP) or overnight at 4 °C (antiphospho-p38). The membranes
were then washed in TBST and incubated for 1 h with horseradish
peroxidase-conjugated secondary antibodies. The specific signals were
detected using an enhanced chemiluminescence detection system (Amersham
Pharmacia Biotech).
Transient Transfection Assay and X-Gal Staining--
The
construction of mammalian expression vector containing the c-Jun
transcriptional activation mutant TAM-67 was done as described in Ref.
38. The pcDNA3 expression vector containing the cDNA insert
encoding an enzymatically inactive form of SEK1 (SEK-AL) was a kind
gift of Dr. J. R. Woodgett (Ontario Cancer Institute, Canada).
MKP-1 plasmid was kindly provided by Dr. N. Tonks (Cold Spring Harbor, NY).
Cells were seeded at 1.5 × 105 cells/well in six-well
plates. After an overnight culture, 4 µg of SEK-AL, TAM-67, or MKP-1 expression vector and 1 µg of a
-galactosidase expression vector (pUT651) were co-transfected using the FuGene6 transfection reagent (Boehringer Mannheim) according to the manufacturer's protocol. After
20 h of transient transfection, cells were rinsed twice with
phosphate-buffered saline and then incubated in complete medium with or
without 125 nM hypericin for 16 h at 37 °C in the dark. Following this period of recovery, the cells were irradiated as
described above. In order to assess the role of p38 MAPK, transfected cells were pretreated with PD169316 1 h before irradiation. Cells were harvested 10 h post-irradiation, washed, and fixed in 1% (v/v) formaldehyde solution in phosphate-buffered saline for 10 min at
room temperature. To identify
-galactosidase enzyme activity, the
fixed cells were washed twice with phosphate-buffered saline and
stained in buffer containing X-gal (1 mg/ml X-gal in 10 mM Na3PO4, pH 7, 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6·3H2O, 2 mM
MgCl2, 0.02% Nonidet P-40, and 0.01% SDS) for 2-6 h at
37 °C. The blue-colored
-galactosidase-expressing cells were
examined with a light microscope. Cell survival was determined by
calculating the percentage of morphologically apoptotic blue cells in
the total number of blue cells in at least ten randomly selected fields.
 |
RESULTS |
Effect of Hypericin on Different MAPK Signaling Pathways--
We
initially investigated whether one or more members of the MAPK family
were activated following cell photosensitization with hypericin (PDT).
In order to assess the general validity of the hypericin-induced
effects on MAPK activities, different human cancer cell lines (A431,
HaCaT, and HeLa cells) or murine L929 cells were incubated for 16 h with concentrations of hypericin that, after exposure to a light dose
of 4 J/cm2, result in 20% cytotoxicity as determined by
the neutral red assay (29). After PDT, cells were further incubated in
the dark and harvested after the specified period of time. Fig.
1 shows that in all cell lines tested,
sublethal doses of PDT with hypericin caused a rapid and persistent
activation of JNK1 while severely inhibiting the basal levels of ERK2
activity. Hypericin without photo-activation had no effect on the
activity of either kinase (Fig. 1, lane 2 in all panels).
These observations strongly suggest that the changes in the activities
of JNK1 and ERK2 are typical light-dependent,
hypericin-mediated responses that are not restricted to a particular
cell line. As shown for HeLa cells in Fig. 1, no difference in the JNK1
or ERK2 protein level was observed over the time period examined,
indicating that the kinase activity changes reflect modifications of
pre-existing molecules.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of photo-activated hypericin on the
activation of ERK2 and JNK1. Cells were incubated for 16 h
with 66 nM (A431), 72 nM (L929), 45 nM (HaCaT) and 81 nM (HeLa) hypericin and were
harvested at the indicated time points after irradiation. JNK1
(A) and ERK2 (B) activities were analyzed in an
immunocomplex kinase assays using GST-c-Jun or MBP as substrates,
respectively as described under "Experimental Procedures." Results
shown are representative of at least three independent experiments.
Equal amounts of protein from HeLa cell lysates were analyzed for
the level of JNK1 or ERK2 proteins by immunoblotting with the specific
antibodies. For both panels, controls of cells untreated (1st
lane) or treated with hypericin without light (2nd
lane) are shown.
|
|
To test whether the inhibition of ERK2 by PDT could be counteracted by
the addition of inducing agents of the ERK2 pathway, we evaluated the
effect of EGF and FCS on PDT-induced ERK2 inhibition. We observed that
in addition to inhibiting the basal cellular levels of ERK2 activity,
photo-activated hypericin blocked the EGF-mediated ERK2 activation
(Fig. 2). The inhibition of the ERK2 pathway was irreversible, as the addition of EGF either 5 min before
(lane 5) or immediately after (lane 6) light
exposure could not prevent or reverse the ERK2 inhibition. Similar
results were obtained when the signal to ERK2 activation was initiated
by the addition of FCS to the cell culture (data not shown). Because a
sustained activation of JNKs, with a concomitant inhibition of ERKs,
has been reported to occur during the induction of apoptosis (16), we
looked next for a correlation between the hypericin-induced effects on
MAPK activities and cell death.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
PDT induces a specific and irreversible
inhibition of the EGF-mediated ERK2 activation. HaCaT cells were
incubated for 16 h with 45 nM hypericin (lanes
2 and 4-7) and then either directly irradiated
(lane 2) or treated with 100 ng/ml EGF 5 min before
(lane 5) or immediately after (lane 6)
illumination. Cells treated with 100 ng/ml EGF for 5 min (lanes
3 and 4) or 20 min (lanes 7 and
8) but not irradiated are shown. ERK2 activity was
determined as described above.
|
|
PDT with Hypericin Induces Sustained JNK1 and p38 MAPK Activations
and Leads to Caspase-mediated PARP Fragmentation and Apoptosis in HeLa
Cells--
In a separate study undertaken to characterize the type of
cell death induced by photo-activated hypericin (39), we have shown
that this photosensitizer can induce either apoptosis or necrosis in
HeLa cells, depending on the photodynamic conditions used. A 16-h
preincubation with 125-250 nM hypericin followed by
irradiation with a light dose of 4 J/cm2 was found to
efficiently and specifically trigger the apoptotic program in this cell line.
Here we show that upon Hoechst 33342 staining and fluorescence
microscopic analysis, the nuclei of cells treated with photo-activated hypericin appeared highly condensed and fragmented in contrast to the
homogeneously stained nuclei of untreated cells (Fig.
3A). Cells treated with
hypericin but not irradiated were indistinguishable from untreated
control cells (data not shown).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3.
PDT with hypericin induces persistent JNK1
and p38 MAPK activation, DEVD-directed caspase activation, and
apoptosis. HeLa cells were incubated for 16 h with 125 nM hypericin and then irradiated as described.
A, nuclear staining with Hoechst 33342 at 24 h
post-irradiation of (panel 1) untreated and (panel
2) PDT-treated cells. B, JNK and p38 MAPK activations
were determined after harvesting the cells at the indicated time points
either by immunocomplex kinase assay (JNK1) or by Western blot using
phospho-p38 antibody. Fold activation was determined for JNK1 by
quantifying the level of GST-Jun phosphorylation by liquid
scintillation counting and for p38 MAPK by scanning the bands for
densitometric analysis. C, PDT-induced PARP cleavage was
determined in cell lysates by Western blot analysis at the indicated
time points post-irradiation (p.i.) as described under
"Experimental Procedures." Controls of cells untreated (lane
1) or treated with hypericin without light (lane 2) are
shown. D, aliquots (50 µg of protein) of cell
lysates prepared at the indicated times after PDT were incubated with
50 µM DEVD-amc at 37 °C for 30 min. The release of amc
was then monitored by a spectrofluorometer with an excitation
wavelength of 360 nm and an emission wavelength of 460 nm. Lysates of
serum-starved (36 h) HeLa cells (S.F.) were taken as a
positive control. n.i. = not irradiated.
|
|
To further characterize the signaling pathways involved in the
hypericin-induced cellular responses, we focused on HeLa cells as a
model system. As shown in Fig. 3B, treatment of the cells with 125 nM hypericin and a light dose of 4 J/cm2 caused a sustained activation of both JNK1 and p38
MAPK over a 24-h time period. While the p38 MAPK activity showed a
steady increase after 1 h post-irradiation and remained sustained
during the time period examined, the activation of JNK1 was also
sustained and somehow biphasic. These JNK1 and p38 MAPK activity
changes resulted from post-translational modifications, since no
difference in protein levels was detected by Western blot analysis over
the 24-h time course (data not shown).
Caspases, a group of cysteine proteases that cleave protein substrates
after aspartic acids, play a central role in the regulation and
execution of the apoptotic program (reviewed in Ref. 40), and the
cleavage of poly(ADP-ribose) polymerase (PARP) by activated caspase-3
subfamily members is considered to be one of the hallmarks of apoptosis
(41). We therefore examined whether photo-activated hypericin could
trigger this event in HeLa cells. As shown in Fig. 3C,
lysates of PDT-treated cells contained the characteristic 85 kDa PARP
fragment, an indication of the activation of the effector caspase-3
and/or caspase-7 (42) and apoptotic cell death. The apoptotic PARP
fragmentation coincided well with the activation of a DEVD-directed
caspase activity (Fig. 3D). While the initial PDT-mediated
induction of the stress kinase pathways clearly preceded the apoptotic
events (Fig. 3B), the sustained activation of p38 MAPK and
the late phase of JNK1 activity continued in parallel with the kinetics
of PARP cleavage and the DEVD-amc proteolytic activity (Fig. 3,
C and D). This observation may suggest a role for
these kinases in the hypericin-induced apoptosis.
Impact of Caspase Inhibitors on the Hypericin-induced JNK1 and p38
MAPK Activation and Apoptosis--
Because a functional cross-talk
between the JNK/p38 MAPK and the caspase signaling pathway has been
reported (43-46), we tested the effect of different caspase inhibitors
on hypericin-induced JNK1 and p38 MAPK activations and apoptosis. HeLa
cells, incubated for 16 h with 125 nM hypericin, were
pretreated for 2 h before irradiation with the caspase-3-directed
inhibitor zDEVD-fmk. As shown in Fig.
4A (lower panel),
preincubation with zDEVD-fmk did not notably affect the PDT-induced
JNK1 or p38 MAPK activations. This caspase inhibitor did not inhibit
the JNK1/p38 MAPK activation during the entire time course examined
(data not shown). These observations indicate that
zDEVD-fmk-inhibitable caspases are not required for the activation of
the JNK/p38 MAPK signaling pathways. On the other hand, zDEVD-fmk
completely blocked the PDT-induced PARP cleavage (Fig. 4A,
upper panel), the DEVD-directed caspase activity and
efficiently counteracted the apoptotic cell death (Fig. 4A,
lower panel). Similar results were obtained when cells were
pretreated with the broad spectrum caspase inhibitor zVAD-fmk (data not
shown). These results demonstrate that while the zDEVD-fmk-inhibitable
caspases are crucial mediators of hypericin-induced cell death, caspase
activation is either unrelated to or lies downstream from the JNK and
p38 MAPK pathways. Furthermore, these observations show that JNK1 and
p38 MAPK are activated whether or not cells will ultimately undergo
apoptosis following PDT.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of caspase inhibitors on PDT-induced
JNK1 or p38 MAPK activations, PARP cleavage, DEVD-caspase activation
and apoptosis in HeLa cells. A, HeLa cells were
incubated for 16 h with 125 nM hypericin and then
pretreated with 100 µM zDEVD-fmk for 2 h before
irradiation. Cells were harvested and analyzed after 10 h (for
JNK1, p38 MAPK, and caspase activities) or 24 h (for PARP cleavage
and apoptosis) post-irradiation. B, time course of
PDT-induced JNK1 and p38 MAPK activations in HeLa cells stably
transfected with the caspase inhibitor CrmA (HeLa-CrmA) or the empty
vector (HeLa-Hyg). Cells were incubated for 16 h with 250 nM hypericin and irradiated. JNK1 and p38 activities were
determined at the indicated time post-irradiation as described above.
C, parental, HeLa-CrmA, and HeLa-Hyg cells were either
exposed to PDT with 250 nM hypericin or incubated with
104 IU/ml TNF in the presence of 1 µg/ml cycloheximide.
PARP cleavage was analyzed as described 16 h after
treatment.
|
|
We also investigated the potential role of caspase-1 and caspase-8 in
the process of hypericin-induced JNK1/p38 MAPK activation and
apoptosis. For this, we used HeLa cells that stably overexpress the
serpin-like protease inhibitor CrmA, which is a selective inhibitor of
caspase-1 and caspase-8 (47). HeLa cells stably expressing the empty
vector (HeLa-Hyg) were taken as control. Fig. 4B shows that
PDT-induced activation of the JNK1 and p38 MAPK was not affected by the
CrmA over-expression. For reasons not clear to us, the CrmA expression
had a stimulatory effect on JNK1 activation at early time points (Fig.
4B), and a similar effect was also observed in parental HeLa
cells treated with the peptide caspase inhibitors (data not shown).
Similar observations have also been reported by others (46) in
different cell lines. Nevertheless, our results clearly indicate that
JNK1 activation in PDT-treated HeLa cells occurs independently from
caspase activities. Moreover, hypericin-induced PARP cleavage was not
blocked in the CrmA-expressing HeLa cells, while the TNF-mediated PARP
fragmentation was completely prevented in the same cell line (Fig.
4C). HeLa-Hyg cells behaved as the parental cells (Fig. 4,
B and C). In agreement with the differential PARP
cleavage results, PDT treatment with hypericin induced a DEVD-directed
caspase activation and apoptosis in the CrmA-expressing HeLa cells,
while the TNF-induced apoptosis in the same cell line was blocked (data
not shown). Altogether, these results indicate that photo-activated
hypericin induces JNK1 and p38 MAPK activation as well as apoptosis in
HeLa cells via a caspase-1- and caspase-8-independent mechanism.
Effect of Blocking the JNK and p38 MAPK Signal Transduction
Pathways on the Hypericin-induced Programmed Cell Death--
The
results presented so far indicate that while the zDEVD-fmk-sensitive
executioner caspases play a critical role in the PDT-induced cell
death, the early CrmA-inhibitable caspases are not essential in this
process. It can also be concluded that the activation of JNK and p38
MAPK signaling pathways do not require these caspase activities.
Therefore, we looked for a possible functional role of JNK1 in the
hypericin-induced apoptosis by transiently transfecting HeLa cells with
dominant negative mutants of SEK1 (SEK-AL) and c-Jun (TAM-67) as
upstream or downstream inhibitors of the JNK signaling pathway,
respectively (2). SEK-AL was generated by mutating the phosphorylation
and activation sites S220 and T224 of the wild type SEK1 into alanine
and leucine, respectively (6). TAM-67 is a c-Jun mutant in which amino
acids 3-122 have been deleted (38). This mutant protein retains the
DNA binding and the leucine zipper domains but lacks most of the
transactivation domain, which contains the phospho-acceptor sites for
JNK and therefore fails to activate the transcription of AP-1
responsive genes. The role of p38 MAPK was assessed by using its
specific pharmacological inhibitor, PD169316 (48). The ectopic
expression of the MAPK phosphatase-1 (MKP-1), which can dephosphorylate
and inactivate both JNK and p38 MAPKs (49), was also used to analyze the importance of these protein kinases in the apoptotic process. Cells
were co-transfected with either TAM-67, SEK-AL, MKP-1 or treated with
the p38 MAPK inhibitor (PD169316) in the presence of the
-galactosidase reporter plasmid pUT651 and then photosensitized with
hypericin. Apoptotic cell death was evaluated 10 h after irradiation by morphological criteria. Fig.
5 shows that PDT treatment with hypericin
in the presence of the empty vector alone resulted in about 45%
apoptotic cells. Inhibition of the JNK pathway by transfecting the
cells with either SEK-AL or TAM-67 increased the number of
apoptotic cells by approximately 25%. Co-transfection of SEK-AL
and TAM-67 rendered the cells even more susceptible to the cell death
and increased hypericin-induced photocytotoxicity by approximately 40%
over control. This oversensitization to PDT-induced apoptosis was
slightly more pronounced when both kinases were simultaneously
inhibited by treating the cells with PD169316 after transfection with
either TAM-67 or SEK-AL. Cells treated with the p38 MAPK inhibitor
alone or in combination with hypericin without light were
indistinguishable from untreated control cells. These results point to
a role for both the JNK1 and p38 MAPK signaling cascades in protecting
the cells from hypericin-induced apoptosis.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of the JNK1 and/or p38 MAPK
pathways sensitizes cells to PDT-induced apoptosis. HeLa cells
were co-transfected with the pUT651 reporter plasmid and the different
expression vectors followed by incubation with 125 nM
hypericin for 16 h as described under "Experimental
Procedures." The empty vector was used to equalize the total
transfected DNA. Where indicated, cells were pretreated with 25 µM PD169316 for 1 h before irradiation. 10 h
after PDT treatment cells were stained with X-gal, and the number of
morphologically apoptotic blue cells relative to the total number of
blue cells in at least 10 randomly chosen fields (× 40) was
determined. The data represent averages of three separate transfection
experiments.
|
|
To substantiate the observation that these stress kinase pathways
protect cells from hypericin-induced apoptosis or at least delay this
process, the kinetics of PARP cleavage and of the DEVD-directed caspase
activity following PDT were evaluated in HeLa cells pretreated with the
specific p38 MAPK inhibitor. As shown in Fig.
6A, in the PD169316 pretreated
cells, PARP cleavage occurred considerably faster than in control
cells. Apoptotic PARP fragmentation was nearly complete 6 h after
irradiation in cells pretreated with the p38 MAPK inhibitor, while the
cleaved 85-kDa product just began to appear in control cells at the
same time period. Similarly, the kinetics of the DEVD-directed caspase
activation were accelerated by cell pretreatment with the p38 MAPK
inhibitor (Fig. 6B). In agreement with these observations,
PD169316 pretreatment increased the potency of the PDT cytotoxic effect
thereby making the cells oversensitive even to sublethal doses of
hypericin (Fig. 6C).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of p38 MAPK inhibition on the kinetics
of PDT-induced PARP cleavage, caspase-3 activation and apoptosis.
HeLa cells were treated with 125 nM hypericin in the
presence or the absence of 25 µM PD169316 as described in
the legend of Fig. 5. Following PDT cells were harvested at the
indicated time points and then analyzed for PARP fragmentation
(A) or DEVD-directed caspase activity (B) as in
Fig. 3. C, cells were treated with the indicated
concentrations of hypericin in the presence or absence of 25 µM PD169316. 10 h after PDT the percentage of cells
with apoptotic morphology in the total number of cells was determined
in at least 10 randomly selected fields (× 40).
|
|
Taken together, these data suggest that the hypericin-induced caspase
and JNK/p38 MAPK activations are part of two functionally distinct and
opposite pathways: the caspase-mediated pathway being required for
apoptosis and the stress kinase cascade being involved in cellular
defense against cell death.
 |
DISCUSSION |
This study reports that PDT with hypericin induces a strong and
persistent activation of the JNK and p38 MAPK signaling pathways while
inhibiting ERK2 activity. Most importantly this is the first study that
outlines a protective role for the JNK/p38 MAPK pathways during
PDT-induced apoptosis which is found to be critically dependent on a
DEVD-directed caspase activity.
The effect of photo-activated hypericin on the MAPK activities was
observed in several cell lines of human and mouse origin, indicating
that it is a general cellular response to this PDT treatment (Fig. 1).
In HeLa cells, the activation of JNK1 and p38 MAPK was very rapid and
sustained for at least 24 h post-irradiation (Fig. 3). We have
shown that photo-activated hypericin, under the conditions used,
induces apoptosis in HeLa cells, an observation that has also been
reported by others using different cell lines (34, 35). Our results
further show that following PDT, the JNK1 and p38 MAPK activation
patterns overlap with the kinetics of PARP cleavage, a DEVD-amc
proteolytic activity and the onset of apoptosis (Fig. 3). Recently, PDT
with a benzoporphyrin derivative has been shown to induce JNK1 and p38
MAPK by a mechanism involving reactive oxygen species production in
murine keratinocytes, but the effects of such activations were not
explored (27). Unlike this observation, cell pretreatment with the
antioxidants N-acetylcysteine or butylated hydroxyanisole
did not affect hypericin-induced stress kinase
activations.2 Moreover, in
the aforementioned study, PDT with BDP did not result in an inhibition
of the ERKs which could be stimulated by EGF during photosensitization
(27). In contrast, here we show that PDT with hypericin caused an
inhibition of the ERK pathway which could not be relieved or prevented
by EGF or FCS (pre)treatment. Interestingly, we have reported
previously that hypericin inhibits the EGF receptor tyrosine kinase
activity in vitro in an irreversible and
light-dependent manner (50).
The relevance of the hypericin-mediated irreversible inhibition of the
ERK signal to the process of PDT-induced cytotoxicity is not yet known.
Intriguingly, several studies have suggested that the dynamic balance
between the ERK and the JNK/p38 MAPK activities critically determines
the cellular fate in response to differentiating, proliferating, or
apoptogenic stimuli (16). However, while the role of the ERK signaling
cascade in the processes of cell differentiation and proliferation has
been clearly recognized, the requirement of JNK1 and p38 MAPK
activations in apoptosis remains a matter of controversy. In some
instances JNK and/or p38 MAPK activity is strictly required for
apoptosis to occur (17-19), while in other circumstances these kinases
are unrelated to the process of programmed cell death (21-23). Strong
and persistent activation of JNK and/or p38 MAPKs with a concurrent
inhibition of ERK has been shown to be critical for apoptosis induced
by UV- and
-irradiation as well as by nerve growth factor withdrawal (16, 18). In addition, a cross-talk between the JNK/p38 MAPK and
caspase activation pathways has been reported in several systems (43-46).
These observations prompted us to look into the possible functional
relationship between the JNK1/p38 MAPK pathways and caspase activation
following PDT. Activation of caspases can be achieved through at least
two mechanisms. The best characterized pathway involves the
autoactivation of procaspase-8 following its recruitment by the death
effector domain of the adapter protein FADD (Fas-associated death
domain), which associates to clustered death domains of Fas/CD95 and
TNFR1 (through TRADD, TNF receptor-associated death domain) upon ligand
binding. This results in the caspase-8-mediated proteolytic activation
of downstream effector caspases, such as caspase-3, caspase-7, and
caspase-9, and commitment to cell death (reviewed in Ref. 51). Another
pathway for caspase activation, triggered by many apoptogenic agents,
involves the release of cytochrome c from the mitochondria
into the cytosol (52). Cytosolic cytochrome c induces the
ATP- or dATP-dependent formation of the "apoptosome," a
complex of proteins composed of cytochrome c itself, Apaf-1,
and procaspase-9 (53). The processing and activation of procaspase-9 in
the complex ultimately leads to the activation of procaspase-3 and cell
death (52). This latter mechanism may also contribute to the
Fas-mediated apoptosis by amplifying the effect of caspase-8 on
downstream caspases (54).
Therefore, we used two approaches to discern the relationship between
the JNK/p38 MAPK and the caspase activation pathways. First, we used an
inhibitor of the downstream effector caspases, zDEVD-fmk, and showed
that while it clearly blocked PARP cleavage and the DEVD-amc
proteolytic activity, and significantly counteracted the
hypericin-induced apoptosis, it did not affect either JNK1 or p38 MAPK
activations (Fig. 4). This indicates that stimulation of the JNK and
p38 MAPK signaling cascades, in response to photo-activated hypericin,
does not require a zDEVD-fmk-inhibitable caspase activity. This in turn
implies that caspase activation is either downstream of the JNK1 and
p38 MAPK activations or that it lies on an independent pathway.
However, our results demonstrate that a DEVD-directed caspase, most
likely caspase-3, is a key mediator of the hypericin-induced cell death.
As a second approach, we examined the potential role of the early
upstream caspase-1 and caspase-8 in PDT-induced JNK and p38 MAPK
activations by stably expressing the serpin-like caspase inhibitor CrmA
in HeLa cells. Overexpression of CrmA did not affect either the
PDT-induced PARP cleavage, DEVD-amc proteolytic activity, or the
JNK1/p38 MAPK activations (Fig. 4) and, moreover, did not prevent the
PDT-induced apoptosis. On the contrary, TNF-mediated PARP cleavage
(Fig. 4) and cell death were completely inhibited in HeLa cells that
overexpress CrmA confirming the central role of procaspase-8 in the
cytotoxic response to TNF (55). These observations demonstrate that
CrmA-inhibitable caspases are not required for PDT-induced apoptosis
and clearly dissociate the hypericin-induced cytotoxicity from the
prototype mechanisms that involve cell surface death domain-containing
receptors. As mentioned above, hypericin could lead to the activation
of a DEVD-directed caspases and ultimately to apoptosis through
mitochondrial damage and release of cytochrome c into the
cytosol. Indeed, we have shown that cytochrome c release is
one of the earliest events induced by PDT in hypericin-treated HeLa
cells (39).
Finally, we present several lines of evidence that the activations of
JNK1 and p38 MAPK may be regarded as cellular protective signals
against hypericin-induced apoptosis. First, the expression of a
dominant negative SEK1 mutant (SEK-AL), a direct upstream activator of
JNK, and/or the transfection of the transactivation-deficient mutant of
the c-Jun protein (TAM-67), the major downstream effector substrate of
JNK, enhanced the rate of hypericin-induced cell death. Co-transfection
of both mutant proteins significantly increased the percentage of
apoptotic cells. Second, the specific pharmacological inhibitor of p38
MAPK, PD169316, also had an enhancing effect on the hypericin-induced
cell death. Combined inhibition of JNK1 and p38 MAPK by PD169316
pretreatment of cells transfected with either SEK-AL or TAM-67
augmented the hypericin-induced apoptosis to nearly 90% (Fig. 5).
Third, by expressing the dual specificity phosphatase MKP-1, which
dephosphorylates and inactivates both JNK1 and p38 MAPK in
vivo, we also observed a potentiation of the hypericin-induced
apoptosis in HeLa cells. These observations were further supported by
the findings that in the presence of the p38 MAPK inhibitor, PDT with
hypericin induced PARP cleavage, DEVD-directed caspase activation, and
apoptosis with much faster kinetics. PD169316 pretreatment also highly
oversensitized the cells to PDT with sublethal doses of hypericin (Fig.
6). Altogether, the results suggest that JNK and p38 MAPK pathways are
required for survival in response to PDT with hypericin. To some
extent, our conclusions are in agreement with the results of a recent study on the protective role of the stress kinase pathways in the
TNF/Fas-induced apoptosis in the murine cell line L929 (56). In that
study, expression of dominant negative mutants of MKK4/SEK1, MKK6 or
TRAF2-DN (all of which are mediators of JNK or p38 activation) enhanced
the TNF-induced apoptosis. Previous studies have also shown that TNF
can induce NF-
B but not JNK in cells from TRAF2-deficient mice and
these cells are more susceptible to apoptosis than those from wild type
mice (57, 58). These reports suggest that JNK and p38 MAPK pathways may
be essential for cell survival in the presence of TNF.
Overall, our results indicate that although JNK1 and p38 MAPK may play
a role in protecting HeLa cells from PDT-induced cytotoxicity, they
cannot rescue cells from hypericin-induced cell death. Most likely they
delay the apoptotic process by inducing the transcription of
survival-promoting genes until a critical threshold of damage finally
commits the cells to apoptosis. An intriguing explanation for the lack
of full protection by the JNK/p38 MAPK pathways could reside in the
concurrent irreversible inhibition of the ERK pathway. It is tempting
to speculate that the JNK/p38 MAPK survival pathway cannot cope with
the hypericin-induced death signal as it is not complemented with an
ERK-mediated proliferation pathway. This could be one of the reasons
why PDT with hypericin has been shown to be a powerful in
vivo anticancer tool. The observation that JNK and p38 MAPK
inhibition enhances the PDT-induced cell death may offer molecular
bases for the development of new therapeutic strategies to enhance the
effectiveness of hypericin as an anticancer tool in PDT.
 |
ACKNOWLEDGEMENTS |
We thank H. De Wulf and G. Nijs for expert
technical assistance, Dr. J. Woodgett for providing the SEK-AL
construct, Dr. N. Tonks for the MKP-1 vector, and Dr. D. Pickup for the
CrmA cDNA.
 |
FOOTNOTES |
*
This work was supported by "Interuniversitaire
Attractiepolen" P4/26, Grant 9005097N of the "Fonds voor
Wetenschappelijk Onderzoek-Vlaanderen," and European Biomed Program
Grant BMH4-CT96-0300.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of a fellowship from the "Vlaams Instituut voor de
Bevordering van het Wetenschappelijk-technologisch Onderzoek in de Industrie."
**
Postdoctoral Researcher with the "Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen."

Research Director with the "Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen."
§§
Research leader with the "Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen". To whom correspondence should be addressed:
Division of Biochemistry, Faculty of Medicine, KULeuven, Herestraat 49 B-3000 Leuven. Tel.: 32-16-345-715; Fax: 32-16-345-995; E-mail: Patricia.Agostinis{at}med.kuleuven.ac.be.
2
Z. Assefa, A. Vantieghem, and P. Agostinis,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein
kinase;
MKK, MAPK kinase;
SEK1, SAPK/ERK kinase;
MKP-1, MAPK
phosphatase-1;
MAPKAP, MAPK-activated protein;
MEKK, MAPK/ERK kinase
kinase;
zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone;
zDEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone;
amc, amino-4-methyl-coumarin;
CrmA, cytokine response modifier A;
PARP, poly(ADP-ribose) polymerase;
TNF, tumor necrosis factor-
;
MOPS, 4-morpholinepropanesulfonic acid;
PDT, photodynamic therapy;
GST, glutathione S-transferase;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside;
FCS, fetal calf serum;
EGF, epidermal growth factor.
 |
REFERENCES |
-
Davis, A. J.
(1994)
Trends Biochem. Sci.
19,
470-473[CrossRef][Medline]
[Order article via Infotrieve]
-
Waskiewicz, A. J.,
and Cooper, J. A.
(1995)
Curr. Opin. Cell Biol.
7,
798-805[CrossRef][Medline]
[Order article via Infotrieve]
-
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556[Free Full Text]
-
Hibi, M.,
Lin, A.,
Smeal, T.,
Minden, A.,
and Karin, M.
(1993)
Genes Dev.
7,
2135-2148[Abstract]
-
Han, J.,
Lee, J.-D.,
Bibbs, L.,
and Ulevitch, R. J.
(1994)
Science
256,
808-811
-
Yan, M.,
Dai, T.,
Deak, J. C.,
Kyriakis, J. M.,
Zon, L. I.,
Woodgett, J. R.,
and Templeton, D. J.
(1994)
Nature
372,
798-800[Medline]
[Order article via Infotrieve]
-
Tournier, C.,
Whitmarch, A. J.,
Cavanagh, J.,
Barret, T.,
and Davis, R. J.
(1997)
Pro. Natl. Acad. Sci. U. S. A.
94,
7337-7342[Abstract/Free Full Text]
-
Moriguchi, T.,
Kuronayagi, N.,
Yamaguchi, K.,
Gotoh, Y.,
Irie, K.,
Kano, T.,
Shirakbe, K.,
Muro, Y.,
Shibuya, H.,
Matsumoto, K.,
Nishida, E.,
and Hagiwara, M.
(1996)
J. Biol. Chem.
271,
13675-13679[Abstract/Free Full Text]
-
Marias, R.,
Wynne, J.,
and Treisman, R.
(1993)
Cell
73,
381-393[Medline]
[Order article via Infotrieve]
-
Gupta, S.,
Campbell, D.,
Dérijard, B.,
and Davis, R. J.
(1995)
Science
267,
389-393[Medline]
[Order article via Infotrieve]
-
Raingeaud, J.,
Whitmarch, A. J.,
Barret, T.,
and Dérijard, B.
(1996)
Mol. Cell. Biol.
16,
1247-1255[Abstract]
-
Rouse, J.,
Cohen, P.,
Trigon, S.,
Morage, M.,
Alonso-Llamazares, A.,
Zamanillo, D.,
Hunt, T.,
and Nebreda, A. R.
(1994)
Cell
78,
1027-1037[Medline]
[Order article via Infotrieve]
-
Mclaughlin, M. M.,
Kumar, S.,
McDonnell, P. C.,
Van Horn, S.,
Lee, J. C.,
Livi, G. P.,
and Young, P. R.
(1996)
J. Biol. Chem.
271,
8488-8492[Abstract/Free Full Text]
-
Johnson, N. L.,
Gardner, A. M.,
Diener, K. M.,
Lange-Carter, C. A.,
Gleavy, J.,
Jarpe, M. B.,
Minden, A.,
Karin, M.,
Zon, L. I.,
and Johnson, G. L.
(1996)
J. Biol. Chem.
271,
3229-3237[Abstract/Free Full Text]
-
Huang, S.,
Jiang, Y.,
Li, Z.,
Nishida, E.,
Mathias, P.,
Lin, S.,
Ulvitch, R. J.,
Nemerow, G. R.,
and Han, J.
(1997)
Immunity
6,
739-749[Medline]
[Order article via Infotrieve]
-
Xia, Z.,
Dickens, M.,
Raingeaud, J.,
Davis, R. J.,
and Greenberg, M. E.
(1995)
Science
270,
1326-1331[Abstract]
-
Verheij, M.,
Bose, A.,
Lin, X. H.,
Yao, B.,
Jarvis, W. D.,
Grant, S.,
Birrer, M. J.,
Szabo, E.,
Zon, L. I.,
Kyriakis, J. M.,
Haimovitz-Friedman, A.,
Fuks, Z.,
and Kolesnick, R. N.
(1996)
Nature
380,
75-79[CrossRef][Medline]
[Order article via Infotrieve]
-
Chen, Y.-R.,
Wang, X.,
Templeton, D.,
Davis, R. J.,
and Tan, T.-H.
(1996)
J. Biol. Chem.
271,
31929-31936[Abstract/Free Full Text]
-
Osborn, M. T.,
and Chambers, T. C.
(1996)
J. Biol. Chem.
271,
30950-30955[Abstract/Free Full Text]
-
Yang, X.,
Khosravi-Far, R.,
Chang, H. Y.,
and Baltimore, D.
(1997)
Cell
89,
1067-1076[Medline]
[Order article via Infotrieve]
-
Liu, Z.-G.,
Hsu, H.,
Goeddel, D. V.,
and Karin, M.
(1996)
Cell
87,
565-576[Medline]
[Order article via Infotrieve]
-
Lenczowski, J. M.,
Dominguez, Z.,
Eder, A. M.,
King, L. B.,
Zacharchuk, C. M.,
and Ashwell, J. D.
(1997)
Mol. Cell. Biol.
17,
170-181[Abstract]
-
Khwaja, A.,
and Downward, J.
(1997)
J. Cell Biol.
139,
1017-1023[Abstract/Free Full Text]
-
Nishina, H.,
Fischer, K. D.,
Radvanyi, L.,
Shahinian, A.,
Hakem, R.,
Rubie, E. A.,
Bernstein, A.,
Mak, T. W.,
Woodgett, J. R.,
and Penninger, J. M.
(1997)
Nature
385,
350-353[CrossRef][Medline]
[Order article via Infotrieve]
-
Dougherty, T. J.,
and Marcus, S. L.
(1992)
Eur. J. Cancer
28,
1734-1742[CrossRef]
-
Kick, G.,
Messer, G.,
Plewig, G.,
Kind, P.,
and Goetz, A. E.
(1996)
Br. J. Cancer
74,
30-36[Medline]
[Order article via Infotrieve]
-
Tao, J.,
Sanghera, J. S.,
Pelech, S. L.,
Wong, G.,
and Levy, J. G.
(1996)
J. Biol. Chem.
271,
27107-27115[Abstract/Free Full Text]
-
Klotz, L. O.,
Fritsch, C.,
Briviba, K.,
Tsacmacidis, N.,
Schliess, F.,
and Sies, H.
(1998)
Cancer Res.
58,
4297-4300[Abstract]
-
Vandenbogaerde, A. L.,
Cuveele, J. F.,
Proot, P.,
Himpens, B. E.,
Merlevede, W. J.,
and de Witte, P. A.
(1997)
J. Photochem. Photobiol. B Biol.
38,
136-142[CrossRef][Medline]
[Order article via Infotrieve]
-
Vandenbogaerde, A. L.,
Geboes, K. R.,
Cuveele, J. F.,
Agostinis, P. M.,
Merlevede, W. J.,
and de Witte, P. A.
(1996)
Anticancer Res.
16,
1611-1618[Medline]
[Order article via Infotrieve]
-
Alecu, M.,
Ursaciuc, C.,
Halalau, F.,
Coman, G.,
Merlevede, W.,
Waelkens, E.,
and de Witte, P.
(1998)
Anticancer Res.
18,
4651-4654[Medline]
[Order article via Infotrieve]
-
Fox, F. E.,
Niu, Z.,
Tobia, A.,
and Rook, A. H.
(1998)
J. Invest. Dermatol.
111,
327-332[Abstract]
-
Weller, M.,
Trepel, M.,
Grimmel, C.,
Schabet, M.,
Bremen, D.,
Krajewski, S.,
and Reed, J. C.
(1997)
Neurol. Res.
19,
459-470[Medline]
[Order article via Infotrieve]
-
Zhang, W.,
Lawa, R. E.,
Hinton, D. R.,
Su, Y.,
and Couldwell, W. T.
(1995)
Cancer Lett.
96,
31-35[CrossRef][Medline]
[Order article via Infotrieve]
-
Harris, M. S.,
Sakamoto, T.,
Kimura,
He, S.,
Spee, C.,
Gopalakrishna, R.,
Gundimeda, U.,
Yoo, J. S.,
Hinton, D.,
and Ryan, S. J.
(1996)
Curr. Eye Res.
15,
255-262[Medline]
[Order article via Infotrieve]
-
Assefa, Z.,
Garmyn, M.,
Bouillon, R.,
Merlevede, W.,
Vandenheede, J. R.,
and Agostinis, P.
(1997)
J. Invest. Dermatol.
108,
886-891[Abstract]
-
He, J.,
Whitacre, C.,
Xue, L.,
Berger, N. A.,
and Oleinick, N. L.
(1998)
Cancer Res.
58,
940-946[Abstract]
-
Alani, R.,
Brown, P.,
Binétruy, B.,
Dosaka, H.,
Rosenberg, R. K.,
Angel, P.,
Karin, M.,
and Birrer, M. J.
(1991)
Mol. Cell. Biol.
11,
6286-6295[Medline]
[Order article via Infotrieve]
-
Vantieghem, A.,
Assefa, Z.,
Vandenabeele, P.,
Declercq, W.,
Vandenheede, J. R.,
Merlevede, W.,
de Witte, P.,
and Agostinis, P.
(1998)
FEBS Lett.
440,
19-24[CrossRef][Medline]
[Order article via Infotrieve]
-
Thornberry, N. A.,
and Lazebnik, Y.
(1998)
Science
281,
1312-1316[Abstract/Free Full Text]
-
Kafmann, S. H.,
Desnoyers, S.,
Ottaviano, Y.,
Davidson, N. E.,
and Poirier, G. G.
(1993)
Cancer Res.
53,
3976-3985[Abstract]
-
Cohen, G. M.
(1997)
Biochem. J.
326,
1-16[Medline]
[Order article via Infotrieve]
-
Cardone, M. H.,
Salvesen, G. S.,
Widmann, C.,
Johnson, G.,
and Frisch, S. M.
(1997)
Cell
90,
315-323[Medline]
[Order article via Infotrieve]
-
Cahill, M. A.,
Peter, M. E.,
Kischkel, F. C.,
Chinnaiyan, A. M.,
Dixit, V. M.,
Krammer, P. H.,
and Nordheim, A.
(1996)
Oncogene
13,
2087-2096[Medline]
[Order article via Infotrieve]
-
Jou, P.,
Kuo, C. J.,
Reynolds, S. E.,
Konz, R. F.,
Raingeaud, J.,
Davis, R. J.,
Biemann, H.-M.,
and Blenis, J.
(1997)
Mol. Cell. Biol.
17,
24-35[Abstract]
-
Muhlenbeck, F.,
Haas, E.,
Schwenzer, R.,
Schubert, G.,
Grell, M.,
Smith, C.,
Scheurich, P.,
and Wajant, H.
(1998)
J. Biol. Chem.
273,
33091-33098[Abstract/Free Full Text]
-
Zhou, Q.,
Snipas, S.,
Orth, K.,
Muzio, M.,
Dixit, V. M.,
and Salvesen, G. S.
(1997)
J. Biol. Chem.
272,
7797-7800[Abstract/Free Full Text]
-
Kummer, J. L.,
Rao, P. K.,
and Heidenriech, K. A.
(1997)
J. Biol. Chem.
272,
20490-20494[Abstract/Free Full Text]
-
Franklin, C. C.,
and Kraft, A. S.
(1997)
J. Biol. Chem.
272,
16917-16923[Abstract/Free Full Text]
-
Agostinis, P.,
Vandenbogaerde, A.,
Donella-Deana, A.,
Pinna, L. A.,
Lee, K.-T.,
Goris, J.,
Merlevede, W.,
Vandenheede, J. R.,
and de Witte, P.
(1995)
Biochem. Pharmacol.
49,
1615-1622[CrossRef][Medline]
[Order article via Infotrieve]
-
Wallach, D.,
Bolin, M.,
Varfolomeev, E.,
Beyaert, R.,
Vandenabeele, P.,
and Fiers, W.
(1997)
FEBS Lett.
410,
96-106[CrossRef][Medline]
[Order article via Infotrieve]
-
Srinivasula, S. M.,
Ahmad, M.,
Fernandes-Alnemri, T.,
Litwack, G.,
and Alnemri, E. S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14486-14491[Abstract/Free Full Text]
-
Liu, X.,
Kim, C. N.,
Yang, J.,
Jammerson, R.,
and Wang, X.
(1996)
Cell
86,
147-157[Medline]
[Order article via Infotrieve]
-
Kuwana, T.,
Smith, J. J.,
Muzio, M.,
Dixit, V.,
Newmeyer, D. D.,
and Kornbluth, S.
(1998)
J. Biol. Chem.
273,
16589-16594[Abstract/Free Full Text]
-
Dbaido, G. S.,
Perry, D. K.,
Gamard, C. J.,
Platt, R.,
Poirier, G. G.,
Obeid, L. M.,
and Hannun, Y. A.
(1997)
J. Exp. Med.
3,
481-490[CrossRef]
-
Roulston, A.,
Reinhard, C.,
Amiri, P.,
and Williams, L. T.
(1998)
J. Biol. Chem.
273,
10232-10239[Abstract/Free Full Text]
-
Lee, S. Y.,
Reichlin, A.,
Santana, A.,
Sokol, K. A.,
Nussenzweig, M. C.,
and Choi, Y.
(1997)
Immunity
7,
703-713[Medline]
[Order article via Infotrieve]
-
Yeh, W.-C,
Shahinian, A.,
Speiser, D.,
Kraunus, J.,
Billia, F.,
Wakeham, A.,
de la Pompa, J. L.,
Ferrick, D.,
Hum, B.,
Iscove, N.,
Ohashi, P.,
Rothe, M.,
Goeddel, D. V.,
and Mak, T. W.
(1997)
Immunity
7,
715-725[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.