From the Drug Discovery Department,
PharmaMar S. A., Tres Cantos, E-28760 Madrid, Spain and the
§ Instituto de Investigaciones Biomédicas "Alberto
Sols," Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid,
E-28029 Madrid, Spain
Received for publication, January 30, 2002, and in revised form, October 21, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report that AplidinTM,
a novel antitumor agent of marine origin presently undergoing Phase II
clinical trials, induced growth arrest and apoptosis in human
MDA-MB-231 breast cancer cells at nanomolar concentrations.
AplidinTM induced a specific cellular stress response
program, including sustained activation of the epidermal growth factor
receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the
serine/threonine kinases JNK and p38 MAPK.
AplidinTM-induced apoptosis was only partially blocked by
the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl
ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an
Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in
MDA-MB-231 cells. Supporting a role for EGFR in AplidinTM
action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also
showed delayed JNK and reduced p38 MAPK activation.
N-Acetylcysteine and ebselen (but not other antioxidants
such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and
vitamin E) reduced EGFR activation by AplidinTM.
N-Acetylcysteine and PP2 also partially inhibited JNK and
p38 MAPK activation. The intracellular level of GSH affected
AplidinTM action; pretreatment of cells with GSH or
N-acetylcysteine inhibited, whereas GSH depletion caused,
hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably,
AplidinTM also induced apoptosis and activated EGFR, JNK,
and p38 MAPK in two cell lines (A-498 and ACHN) derived from human
renal cancer, a neoplasia that is highly refractory to chemotherapy.
These data provide a molecular basis for the anticancer activity of
AplidinTM.
AplidinTM (a cyclic depsipeptide,
C57H89N7O15,
Mr = 1112) is a novel antitumor agent isolated
in 1990 from the Mediterranean tunicate Aplidium albicans
(1). In vitro, AplidinTM has potent cytotoxic
activity against breast, colon, non-small cell lung, and prostate
carcinoma and melanoma cells (2, 3). It has also shown strong antitumor
activity in various xenograft models (4). AplidinTM has
recently entered Phase II clinical trials in a variety of solid tumors
upon showing promising toxicity and pharmacological properties in Phase
I studies (5).
Little is known about the mechanism of action of AplidinTM.
Early studies indicated that it exerts an antiproliferative effect through the inhibition of protein synthesis and reduction of ornithine decarboxylase activity (6, 7). In addition, AplidinTM
inhibits vascular endothelial factor secretion and autocrine stimulation in a human leukemia cell line (8).
Many anticancer drugs elicit apoptosis in cancer cells (9-11). Several
studies have reported the activation of one or more members of the
MAPK1 family of intracellular
signaling kinases by cytotoxic agents. Among them, JNK and p38 MAPK
and, less frequently, ERK1/2 play important roles in these apoptotic
processes (12-18). However, JNK and p38 MAPK are not always involved
in apoptosis. They also promote proliferation, differentiation, or
survival depending on the cell type and external stimulus (19-22).
Interestingly, JNK is also activated by chemopreventive agents such as
the isothiocyanates present in cruciferous vegetables (23) and has been
shown to lie upstream of the caspase proteases that constitute the
effector system in the apoptotic pathway (14). However, in other cases, such as apoptosis induced by doxorubicin or To study the mechanism of action of AplidinTM in human
cancer cells, we chose the MDA-MB-231 breast cell line, which contains mutated p53 and ras genes and is highly invasive and
proliferative. Here we report that AplidinTM induced rapid
and sustained activation of the epidermal growth factor receptor
(EGFR), the non-receptor tyrosine kinase Src, JNK, and p38 MAPK.
SB203580, an inhibitor of both JNK and p38 MAPK in these cells,
substantially inhibited the cytotoxic effect of AplidinTM,
suggesting an important role of these kinases in AplidinTM
action. These effects depend on a decrease in intracellular GSH. In
addition, we found that EGFR activation by AplidinTM was
partially mediated by Src, but was somehow abnormal because it did not
lead to the same binding of Shc and Grb2 adaptor proteins as that
induced by EGF. In view of the promising effects observed in patients
in Phase I studies, we extended the study to two lines, ACHN and A-498
cells (both wild-type p53), of renal cancer, a neoplasia highly
refractory to chemotherapy. In these cells, AplidinTM also
activated EGFR, JNK, and p38 MAPK, but did not affect the high
endogenous level of active Src.
Cell Lines, Antibodies, and Reagents--
MDA-MB-231, ACHN, and
A-498 cells were obtained from American Type Culture Collection
(Manassas, VA) and grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum and 1 mM glutamine
(all from Invitrogen). Wild-type and EGFR-deficient (egfr
Anti-JNK1, anti-p38 MAPK, anti-ERK, anti-human EGFR, anti-Grb2, and
anti-Src antibodies (for Western blotting) were from Santa Cruz
Biotechnology. Anti-Src antibody (for immunoprecipitation) was
generously donated by Dr. J. Brugge. Anti-phospho-JNK1,
anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-Src
Tyr418, anti-phospho-Akt Ser348, anti-caspase-3
(active form), and anti- Nuclear Staining Assays--
After treatment, cells were washed
once with phosphate-buffered saline (PBS) and fixed in a solution of
chilled methanol/acetic acid (1:1) for 2 min. The fixed cells were
washed with PBS, placed on slides, and stained with 2 µg/ml
4,6-diamidino-2-phenylindole for 15 min. Excess dye was washed off with
PBS. Nuclear morphology was observed under a fluorescence microscope
(Zeiss Axiophot).
Flow Cytometry Analysis--
Cells after different treatments
with AplidinTM and/or inhibitors were stained with
propidium iodide (Sigma) and analyzed by flow cytometry (FACScan, BD
Biosciences). For staining, cells (1 × 106) were
harvested, washed with PBS, and then fixed in 70% ethanol. Fixed cells
were treated with DNase-free RNase for 30 min at 37 °C, washed with
PBS, centrifuged, and incubated in PBS-containing propidium iodide (25 µg/ml). Forward light scatter characteristics were used to exclude
the cell debris from the analysis. Apoptotic cells were determined
by their hypochromic subdiploid staining profiles. To estimate early
apoptotic cells, Alexa 488-conjugated annexin V (Molecular Probes,
Inc.) was used together with propidium iodide dead cell counterstain
following the manufacturer's recommendations.
DNA Synthesis Measurements--
Cells (3.5 × 104) were seeded in 24-well dishes (Nunc). One day later,
cells were treated or not with AplidinTM in normal growth
medium and pulsed with 5 µCi/ml [3H]thymidine (Amersham
Biosciences) for 30 min at the indicated times post-treatment. At the
end of the labeling period, the medium was removed, and the cells were
rinsed twice in PBS and fixed with chilled 10% trichloroacetic acid
for 10 min. Trichloroacetic acid was then removed, and the monolayers
were washed with ethanol and air-dried at room temperature for 20 min.
Thereafter, precipitated macromolecules were dissolved in 500 µl of
0.5 N NaOH and 0.1% SDS, and 450 µl of each sample was
diluted in 5 ml of OptiPhase HighSafe scintillation solution (Wallac).
Radioactivity was measured on a 1209 RackBeta counter (Wallac).
Crystal Violet Staining Method--
To estimate cell mass, the
medium was removed, and 24-well dishes were washed with PBS, fixed with
1% glutaraldehyde for 15 min, washed twice with PBS, and stained with
100 µl of 0.1% aqueous crystal violet for 20 min. Dishes were rinsed
four times in tap water and allowed to dry. One-hundred µl of 10%
acetic acid was added, and the content of each well was mixed before
reading the absorbance at 595 nm.
Immunoprecipitation and Western Blot Analysis--
To study the
effect of AplidinTM on the activity of different kinases
(Src, p38 MAPK, JNK, ERK, and Akt), cells were preincubated for 24 h in serum-free medium. For immunoprecipitation, cells were lysed in
modified radioimmune precipitation assay buffer (50 mM
Hepes (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton
X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM AplidinTM Induces Apoptosis in MDA-MB-231 Breast
Cancer Cells--
AplidinTM showed high activity against
MDA-MB-231 cells (Fig. 1A),
with an antiproliferative IC50 of 5 nM at
48 h post-treatment (data not shown). This concentration is
pharmacologically relevant, as the circulating plasma levels of
AplidinTM in patients exposed to the maximum tolerated dose
in Phase I clinical studies is 7.1 nM (26).
Dose-dependent inhibition of DNA synthesis was detected
1-3 h after drug addition (Fig. 1B). By flow cytometry
analysis, AplidinTM was found to induce the subdiploid
profile, which is a hallmark of apoptosis (Fig. 1C). At
24 h, 20-25% apoptotic cells were found in cultures growing in
normal medium treated with 50-500 nM
AplidinTM, and ~10% apoptotic cells were found in
cultures treated with 5 nM AplidinTM. In line
with this, AplidinTM-treated cells showed time- and
dose-dependent nuclear condensation and fragmentation in
comparison with the homogeneous staining of untreated cells as assessed
by 4,6-diamidino-2-phenylindole staining (Fig. 1D,
upper panels) and the generation of the active form of
caspase-3 (lower panels). When added simultaneously to the
drug, benzyloxycarbonyl-VAD-fluoromethyl ketone, a general caspase
inhibitor, decreased the cytotoxic activity of AplidinTM by
~50% as assessed by crystal violet staining (data not shown). This
indicates that AplidinTM-induced apoptosis is mediated by
caspase-dependent and -independent mechanisms. The
cytotoxic action of AplidinTM is triggered rapidly, as a
pulse treatment of 5 min (using 500 nM) gave the maximum
antiproliferative effect at 48 h (data not shown).
AplidinTM-induced Apoptosis Relies on Sustained JNK and
p38 MAPK Activation--
Doses 10-100-fold higher than the
antiproliferative IC50 were used to identify the primary
targets and early mechanism of action of AplidinTM. To
examine the role of MAPKs in AplidinTM-induced apoptosis,
we studied the effects of AplidinTM on the activity of
ERK1/2, JNK, and p38 MAPK using antibodies specifically recognizing the
active phosphorylated forms. AplidinTM caused rapid,
strong, and sustained activation of p38 MAPK and JNK without altering
their concentrations (Fig.
2A). The same result was
obtained in immune complex kinase assays (data not shown). Addition of
AplidinTM in vitro to immune complexes from
untreated cells did not affect kinase activity (data not shown),
suggesting that p38 MAPK/JNK activation in vivo is due to
effects on their at least 11 upstream regulators (22). In agreement
with the expression of an activated K-ras oncogene (27),
MDA-MB-231 cells displayed a high basal level of phosphorylated ERK1/2
under unstimulated conditions, which was mostly unaffected by
AplidinTM treatment (Fig. 2A). Likewise,
AplidinTM did not change the cellular content of
phosphorylated Akt (Fig. 2A), an enzyme that prevents cell
death by multiple apoptosis inducers (see Ref. 28 for review).
To examine the importance of these effects in the induction of
apoptosis, we used SB203580, a pyridinylimidazole that inhibits some
p38 MAPK isoforms (29, 30). However, control experiments showed that,
in MDA-MB-231 cells, SB203580 inhibited both JNK and p38 MAPK at 5-30
µM (Fig. 2B) (data not shown). This correlated with the inhibition of AplidinTM-induced apoptosis. As
determined by flow cytometry, SB203580 (30 µM) decreased
the number of apoptotic cells from 41 to 16% after 24 h of
AplidinTM treatment in serum-free medium (Fig.
2C), an effect clearly visible by phase-contrast microscopy
(Fig. 2D). These data indicate that the activation of JNK
and/or p38 MAPK plays a critical role in the induction of MDA-MB-231
cell apoptosis by AplidinTM. Moreover, the activation of
these kinases was unaffected by benzyloxycarbonyl-VAD-fluoromethyl
ketone (data not shown), suggesting that these events lie upstream of
caspase activation in the apoptosis cascade.
AplidinTM Induces Src-dependent EGFR
Phosphorylation--
Because JNK activation by some stimuli involves
Src-mediated EGFR phosphorylation (31), we studied whether
AplidinTM could have this mode of action. We found that
AplidinTM caused EGFR phosphorylation, which was partially
blocked by pretreatment with PP2 (Fig.
3A), a specific inhibitor of
Src (32, 33). AplidinTM induced a more sustained EGFR
phosphorylation than EGF (Fig. 3B, upper panels);
it peaked at 1-4 h and remained above the base line for 24 h
(middle panels). Accordingly, the activation-linked down-regulation of EGFR content was delayed in
AplidinTM-treated cells with respect to EGF-treated cells
(Fig. 3B, middle panels). Combined treatment of
MDA-MB-231 cells with EGF and AplidinTM resulted in
cellular toxicity and persistent EGFR phosphorylation, indicating a
dominant and probably aberrant effect of AplidinTM (Fig.
3B, lower panels). This was further suggested by
analysis of the binding of the adaptor proteins Grb2 and Shc to EGFR.
In contrast to what happened upon EGF addition, EGFR activation by AplidinTM did not cause binding of Grb2 or Shc to the
receptor (Fig. 3C). Moreover, in cells treated with both
agents, Grb2 binding was also inhibited, as was binding of Shc at early
times (2 and 30 min post-treatment), although it strongly increased
later (60 min) (Fig. 3C).
EGFR is subjected to inhibitory and activating phosphorylation of
specific residues by several kinases. Five sites of in vivo autophosphorylation have been identified in EGFR: Tyr992,
Tyr1068, Tyr1086, Tyr1148, and
Tyr1173 (34-36). To make sure that AplidinTM
has a stimulatory effect on EGFR, we used residue-specific
anti-phosphotyrosine antibodies. At least four of these five amino
acids became phosphorylated upon AplidinTM addition (Fig.
3D). To assess the specificity of EGFR activation, the
effect of AplidinTM on PDGFR was studied in mouse NIH 3T3
fibroblasts. AplidinTM did not induce phosphorylation of
this receptor (Fig. 3E). These cells were sensitive to
AplidinTM, although they lack EGFR expression, indicating
that EGFR transactivation is not required for
AplidinTM-induced apoptosis. Additionally, given that PP2
significantly reduces AplidinTM-induced EGFR
phosphorylation and that Src transactivates EGFR in some cell systems
(31, 37, 38), we studied the effect of AplidinTM on Src
expression and activity. By means of an antibody that specifically
recognizes the activation-linked phospho-Tyr418 of Src,
AplidinTM rapidly activated Src without altering the amount
of protein (Fig. 3F). Because MDA-MB-231 cells express Src
and Fyn, but not Yes (data not shown), which are the three ubiquitously
expressed members of the Src kinase family that have at least partially overlapping functions (39), the possibility that AplidinTM
may also affect Fyn cannot be ruled out.
The role of EGFR activation in AplidinTM action was studied
in egfr Glutathione Depletion Is Critical for Activation of Src, EGFR, JNK,
and p38 MAPK by AplidinTM--
Reactive oxygen species
mediate JNK activation by pro-inflammatory cytokines (interleukin-1 and
tumor necrosis factor-
We next analyzed the possible link between GSH content, Src and EGFR,
and the activation of JNK and p38 MAPK. Exogenously added GSH and
N-acetylcysteine (but not BSO) reduced JNK and p38 MAPK
activation by AplidinTM (Fig.
6A). Likewise, GSH also
blocked Src activation by AplidinTM, whereas BSO had no
effect (Fig. 6B). In addition, PP2 and tyrphostin AG1478 (an
EGFR inhibitor) also inhibited the activation of these kinases by
AplidinTM (Fig. 6C). Together, these
data indicate that the depletion of GSH activates Src and that the
activation of Src and EGFR is partially responsible for the activation
of JNK and p38 MAPK. EGF alone did not induce p38 MAPK and induced JNK
only weakly and did not affect the activation of these two kinases by
AplidinTM (Fig. 6D).
AplidinTM Induces Cytotoxicity in Renal Cancer
Cells--
To examine whether the mechanism of action of
AplidinTM in MDA-MB-231 cells could be extrapolated to
human renal cancer cells, we used the A-498 and ACHN cell lines. Both
were sensitive to the antiproliferative (IC50 = 5 and 15 nM, respectively) and cytotoxic action of
AplidinTM (Fig.
7A). As in MDA-MB-231 cells,
we found that AplidinTM induced EGFR phosphorylation in
A-498 cells (Fig. 7B). The effect on Src was also
investigated. In contrast to breast cancer cells, both A-498 and ACHN
cells showed a high level of Src activity, which was not affected by
AplidinTM (Fig. 7C). Because the drug activated
JNK and p38 MAPK in A-498 and ACHN cells (Fig. 7D), these
results indicate that, in these two renal cancer cell lines, the
activation of these kinases by AplidinTM does not require
further Src activation. SB203580 reduced the activation of both kinases
(Fig. 7D). The contribution of EGFR, p38 MAPK, and JNK
activation and also of Src to the cytotoxic effect of
AplidinTM in A-498 and ACHN cells was studied by flow
cytometry using AG1478, PP2, and SB203580. In ACHN cells, all three
inhibitors reduced the population of hypodiploid cells with the
following range of potency: SB203580 > AG1478 > PP2 (Table
I). Double AG1478 + SB203580 treatment
was more effective than either agent alone, whereas combined treatments
including PP2 inhibited also AplidinTM, but showed high
toxicity in untreated cells. A possible explanation may be that
inhibition of the basal activity of more than one of the target enzymes
of these agents is deleterious, whereas cells treated with
AplidinTM and combinations of inhibitors may display kinase
activities similar to those of untreated cells. A-498 cells were very
sensitive to the absence of serum (data not shown); and unexpectedly,
in low serum, neither of the inhibitors reduced the induction of apoptosis by AplidinTM (Table I). In these cells, only
the double AG1478 + SB203580 treatment had a small inhibitory effect,
whereas the triple AG1478 + SB203580 + PP2 treatment had a higher
effect, although it was toxic in the absence of
AplidinTM.
We have examined the effect of the novel antitumor agent
AplidinTM in human breast and renal cancer cells.
AplidinTM shows cytotoxicity at pharmacologically relevant
concentrations linked to both inhibition of cell proliferation and
apoptosis. It induces apoptosis via caspase-dependent
and -independent mechanisms irrespective of the cellular p53 status
through the sustained activation of the serine/threonine kinases
JNK and p38 MAPK. These events are triggered rapidly in part by the
induction of GSH depletion and Src tyrosine kinase.
AplidinTM activates EGFR by a mechanism that is partly
dependent on Src activation. Several findings indicate that EGFR
activation by AplidinTM is aberrant. First, it is weaker
and more prolonged than that induced by EGF; second, it is accompanied
by a delay in the down-regulation of the receptor; and third, it does
not induce binding to the receptor of adaptor proteins such as Grb2 and
Shc, suggesting differences in signaling with respect to EGF. Our data
obtained with human MDA-MB-231 and renal ACHN cancer cells and with
mouse fibroblasts indicate that EGFR activation is involved in, but is
not sufficient for, AplidinTM-induced apoptosis. Apart from
its common physiological ligands EGF and transforming growth
factor- Src has been proposed to transactivate EGFR upon several stimuli such
as oxidative stress and G-protein-coupled receptor activation, in the
latter case by forming a complex with Pyk2 that directly phosphorylates
EGFR (38). In other systems, EGFR phosphorylation by Src results in
hyperactivation of receptor kinase activity (37, 43) and in the
enhancement of the mitogenic response of cells to EGF (44). Because Src
binds to and is a substrate of EGFR (45), an activation loop resulting
from this mutual catalytic regulation between both kinases (46) can
amplify the AplidinTM signal causing the abnormally long
activation and half-life of EGFR. To what extent this activation loop
is responsible for the sustained activation of JNK and p38 MAPK is
unknown, but the partial inhibition by tyrphostin AG1478 supports this
idea. Although direct binding of AplidinTM to EGFR or
indirect induction of receptor dimerization cannot be ruled out, our
data support that AplidinTM causes EGFR activation through
the previous activation of Src. In line with our data showing the
activation of Src by AplidinTM, deregulation of Src
activity is involved in the caspase-independent apoptosis induced by
the adenoviral early region 4 open reading frame-4 protein (47).
Thus, like c-Myc (see Ref. 48 for review), c-Src may transduce either
proliferative or apoptotic signals.
AplidinTM action is inhibited by
N-acetylcysteine, a potent antioxidant that can be a source
of sulfhydryl metabolites, stimulate GSH synthesis, enhance glutathione
S-transferase activity, promote detoxification, and act
directly on reactive oxygen species (49). Together with the inhibitory
effects of exogenous GSH and the stimulation by BSO of EGFR and Src
activation by AplidinTM and the activation of Src by
reactive oxygen species in many cell systems, these results strongly
suggest that the activation of Src by AplidinTM depends on
the depletion of cellular GSH. N-Acetylcysteine has been
found to inhibit receptor kinase signaling in many cell systems, suppressing the activation of ERK by H2O2 (50);
of ERK and JNK by interleukin-1 (51); and of ERK, JNK, and p38 MAPK by
arsenite (52). Therefore, AplidinTM might act by inducing
oxidative stress at the plasma membrane, leading to a reduction in GSH
levels and Src activation. Alternatively, AplidinTM may
directly modulate GSH synthesis and/or metabolism. Early GSH depletion
is suggestive of an oxidative process and renders the cells more
sensitive or precedes apoptotic cell death induced by various agents
(Ref. 53 and references therein), although this is not the case for
others such as camptothecin and etoposide (54). GSH regulates JNK and
p38 MAPK (55). Similar to our results, the level of intracellular GSH
is a critical regulator of the induction of JNK and p38 MAPK by
alkylating agents such as methyl methanesulfonate and
N-methyl-N'-nitro-N-nitrosoguanidine (56).
JNK is involved in many cellular responses such as proliferation,
differentiation, and apoptosis. It is thought that the duration of its
activation may be crucial in the signaling decision; sustained (but not
transient) JNK activation participates in the apoptosis induced by
several stress stimuli (12, 57, 58). This indicates that the sustained
JNK activation reported in this work is crucial for the pro-apoptotic
effect of AplidinTM. Like JNK, p38 MAPK is also involved in
the apoptotic response of many cells to cytotoxic agents (16, 59). Our
results show that the activation of both p38 MAPK and JNK by
AplidinTM depends in part on Src activation, as does that
caused by changes in the redox status in some systems (33).
Data obtained using inhibitors indicate that, in human breast cancer
cells, activation of EGFR, Src, and p38 MAPK/JNK is involved in
AplidinTM action, whereas Src activation is dispensable in
MEFs. Compared with breast cells, the inhibitory effect of AG1478, PP2,
and SB203580 is variable in human renal cancer cells (similar in ACHN
cells, but lower in A-498 cells), suggesting that other signaling
pathways may be involved in AplidinTM action in this
cell line. Therefore, AplidinTM-induced apoptosis in
carcinoma cells is partly the result of Src activation, perhaps in
combination with additional effects of GSH depletion, which leads to
the sustained activation of JNK and p38 MAPK. Because
N-acetylcysteine and PP2 inhibit but do not completely block
p38 MAPK/JNK activation by AplidinTM and are cytotoxic in
long incubations, the contribution of GSH depletion and Src activation
cannot be precisely determined, and other upstream regulators of p38
MAPK/JNK might be involved in AplidinTM action. The lack of
appropriate specific inhibitors of JNK or p38 MAPK has hampered the
elucidation of the role of each individual kinase.
Several anticancer drugs in clinical use modulate JNK activity. JNK
mediates the apoptosis induced by DNA-damaging drugs such as etoposide
(VP-16) and camptothecin in human myeloid leukemia cells (14) and of
vinblastine in KB3 carcinoma cells (18). In MDA-MB-231 cells,
paclitaxel-induced apoptosis is mediated by the induction of JNK,
which causes inactivation by phosphorylation of the anti-apoptotic
Bcl-2 protein (17). Taxol has also been shown to increase p38 MAPK,
ERK, and (to lesser extent) JNK activity in human breast cancer cells
(59). Strikingly, tamoxifen, a partial agonist/antagonist of the
estrogen receptor that has a cytostatic action by inhibiting estrogen
action, also has a cytotoxic effect in the estrogen receptor-negative
MDA-MB-231 and BT-20 cell lines via the induction of apoptosis (61).
This effect is mediated by JNK activation and inhibited by vitamin E,
but not by N-acetylcysteine or GSH (60). In HepG2
hepatoma cells, 5-fluorouracil-induced apoptosis is mediated by JNK
activation (61). Also, cisplatin induces apoptosis through both JNK and p38 MAPK (16).
In summary, we report that AplidinTM induces apoptosis in
human breast and renal cancer cells via the sustained activation of the
signaling kinases JNK and p38 MAPK. We show that, in MDA-MB-231 cells,
this is in part a consequence of Src activation, which is preceded by
cellular GSH depletion. In addition, AplidinTM causes
aberrant EGFR activation, which is also involved in the activation of
p38 MAPK and JNK in MDA-MB-231 cells and mouse fibroblasts and in the
cytotoxic effect in the latter and in renal ACHN cells. To establish
the contribution of the abnormal EGFR activation to the anticancer
activity of AplidinTM will require the correlation of EGFR
expression and tumor sensitivity in patients. Our results indicate that
there must be additional AplidinTM targets leading to p38
MAPK/JNK activation. In view of the promising clinical results of
AplidinTM, the identification of these targets and also the
elucidation of the signaling from GSH, EGFR, and Src to JNK and p38
MAPK merit further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation, caspase activation is independent of JNK (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mouse embryo fibroblasts (MEFs) were
obtained from Dr. E. Wagner (Institut für Molekulare Pathologie,
Vienna, Austria). AplidinTM is manufactured by PharmaMar
S. A. (Madrid, Spain). Stock solutions were freshly prepared in
dimethyl sulfoxide and diluted in the cell culture to final
concentrations as indicated. Ascorbic acid, diphenyleneiodonium (DPI),
Tiron, vitamin E ((+)-
-tocopherol acid succinate), catalase,
H2O2, GSH, N-acetylcysteine,
ebselen, L-buthionine (SR)-sulfoximine (BSO),
and 4,6-diamidino-2-phenylindole were from Sigma. PP2
(4-amino- 5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine), tyrphostin AG1478,SB203580, and benzyloxycarbonyl-VAD-fluoromethyl ketone caspase inhibitor were from Calbiochem. Human EGF was from PreproTech Ltd.
-actin antibodies were from New England
Biolabs Inc. Anti-platelet-derived growth factor receptor (PDGFR)
antibody was from Upstate Biotechnology, Inc. Anti-phosphotyrosine and
anti-Shc antibodies were from Transduction Laboratories.
Anti-phospho-EGFR
Tyr1068/Tyr1086/Tyr1148/Tyr1173
antibodies were from BioSource, International.
-glycerophosphate,
100 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and
1 mM phenylmethylsulfonyl fluoride). Lysates were
precleared by incubation with protein G-Sepharose at 4 °C for 30 min
and then incubated overnight with the corresponding antibody (1 µg/ml). After a 1-h incubation with protein G-Sepharose, immune
complexes were washed three times with the same radioimmune precipitation assay buffer lacking deoxycholic acid and SDS and three
times with 50 mM Hepes (pH 7.4), 150 mM NaCl,
10% glycerol, 0.1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM
-glycerophosphate, 100 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride
and electrophoresed on 8% (EGFR and PDGFR) or 12% (Src) acrylamide
gels. For Western blot analysis, cell protein extracts were prepared
following standard procedures (25). Protein extracts were
electrophoresed on 8 or 15% polyacrylamide gels and transferred to
nylon membranes (Immobilon P, Millipore Corp.). The filters were
washed, blocked with 5% bovine serum albumin in Tris-buffered saline
(25 mM Tris (pH 7.4), 136 mM NaCl, 2.6 mM KCl, and 0.5% Tween 20), and incubated overnight at
4 °C with the appropriate antibody (1:1000 dilution). Blots were
washed three times for 10 min with PBS + 0.1% Tween 20 and incubated
with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or
anti-goat antibody for 1 h at room temperature. Blots were
developed by a peroxidase reaction using the ECL detection system
(Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
Effect of AplidinTM on
MDA-MB-231 cell growth and viability. A, phase-contrast
micrographs of cells treated with 50 nM
AplidinTM (APL) or vehicle (Control)
for 24 h. Scale bar = 25 µM.
B, dose-dependent kinetics of the inhibition of
DNA synthesis by AplidinTM. DNA synthesis was calculated by
measuring thymidine incorporation as described under "Experimental
Procedures" at the indicated times after treatment with 5 nM ( ), 50 nM (
), or 500 nM
(
) AplidinTM. C, flow cytometry analysis of
DNA content in cultures incubated in the absence (Control)
or presence of the indicated doses of AplidinTM in normal
growth medium for 24 h. Percentages of apoptotic cells
corresponding to the subdiploid population are shown. D,
upper panels, fluorescence microscope image of cultures
treated with vehicle or AplidinTM (500 nM) for
24 h after nuclear staining with 4,6-diamidino-2-phenylindole.
Arrows indicate cells at an advanced stage of nuclear
condensation and degradation. Lower panels, Western blot
analysis showing the activation of caspase-3 upon AplidinTM
treatment. An antibody that specifically recognizes the active
truncated caspase-3 polypeptide was used. As a control, filters were
reprobed with anti-
-actin antibody.
View larger version (65K):
[in a new window]
Fig. 2.
AplidinTM induces
apoptosis in MDA-MB-231 cells through the sustained activation of p38
MAPK and JNK. A, levels of activated p38 MAPK, JNK,
ERK, and Akt in cells treated with AplidinTM (500 nM) for the indicated times or left untreated (control
(C)) were measured by Western blotting using phospho
(P)-specific p38 MAPK, JNK, ERK, and Akt antibodies.
Antibodies against total protein (p38 and JNK) were used to rule out
effects at the expression level. B, SB203580 inhibited p38
MAPK and JNK activation by AplidinTM. Cells were pretreated
with SB203580 (30 µM) or vehicle for 1 h before
AplidinTM addition as indicated. Western blot analysis was
carried out as described for A. C, the results
from flow cytometry analysis showed that AplidinTM-induced
apoptosis was inhibited by SB203580. Cells were treated with vehicle
(CONTROL), AplidinTM (APL; 500 nM), SB203580 (30 µm) or AplidinTM plus
SB203580 (same doses) for 24 h in serum-free medium. D,
shown are phase-contrast micrographs of cultures treated for 48 h
with vehicle, AplidinTM, SB203580, or AplidinTM
plus SB203580 as described for C. Scale bar = 35 µM. Values shown correspond to a representative
experiment. The experiments shown in A and B were
performed three times, and those in C and D were
performed twice.
View larger version (68K):
[in a new window]
Fig. 3.
AplidinTM induces
Src-dependent EGFR phosphorylation. A,
MDA-MB-231 cells were either pretreated or not for 1 h with PP2
(25 µM) and then treated with EGF (20 ng/ml, 5 min) or
AplidinTM (APL; 500 nM, 1 h) as
indicated. Phosphorylated (P) EGFR levels were analyzed by
immunoprecipitation with anti-EGFR antibody followed by Western
blotting using anti-phosphotyrosine antibody. Total EGFR levels were
measured by reprobing the filters with anti-EGFR antibody.
B, shown is the time course of induction of EGFR
phosphorylation by EGF (20 ng/ml; upper panels),
AplidinTM (500 nM; middle panels),
or both (same doses; lower panels) analyzed as described for
A. C, shown is the time course of Grb2 and Shc
binding to EGFR upon treatment with EGF (20 ng/ml),
AplidinTM (500 nM), or both. Binding was
analyzed by immunoprecipitation (IP) with anti-EGFR antibody
followed by Western blotting (WB) using anti-Grb2 or
anti-Shc antibody. Western blotting using these antibodies was done to
estimate total Grb2 and Shc content. D, cells were treated
for the indicated times with either EGF (20 ng/ml) or
AplidinTM (500 nM). Western blotting (20 µg/lane) was performed using each of the four residue-specific
anti-phosphotyrosine (P-Y) antibodies shown. E,
PDGFR was not phosphorylated upon AplidinTM treatment. NIH
3T3 cells were treated with AplidinTM (500 nM)
or platelet-derived growth factor (PDGF; 10 ng/ml) for the
indicated times. Phosphorylated and total PDGFRs were measured by
immunoprecipitation and Western blotting using anti-PDGFR and
anti-phosphotyrosine antibodies. F, AplidinTM
induced rapid and sustained Src phosphorylation. Cells were treated
with vehicle (control (C)) or AplidinTM (500 nM) for the indicated times, and the amount of
activation-linked phosphorylated Src was measured by
immunoprecipitation with anti-Src antibody followed by Western blotting
using anti-phospho-Tyr418 antibody as described under
"Experimental Procedures." The amount of total Src in the
immunoprecipitates is shown below. Results shown correspond to a
representative experiment. The experiments shown in A and
B were performed three times, and those in C-F
were performed twice.
/
MEFs. In both the presence and
absence of serum, egfr
/
MEFs underwent
apoptosis with delayed kinetics in comparison with wild-type MEFs (Fig.
4A) (data not shown). At late
times after treatment, however, no differences were found in cell
proliferation or viability (data not shown). Consistent with this, the
activation of p38 MAPK and JNK egfr
/
MEFs
was reduced and delayed, respectively, compared with wild-type MEFs
(Fig. 4B, upper panels). To examine the
contribution of EGFR and Src to the activation of p38 MAPK/JNK by
AplidinTM, we used PP2. Pretreatment with PP2 and also with
GSH caused only partial reduction of p38 MAPK and JNK activation in
egfr
/
and wild-type MEFs. SB203580 had a
stronger effect than PP2 and GSH in egfr
/
cells. In addition, we studied the contribution of the activation of
Src and p38 MAPK/JNK to the cytotoxic action of AplidinTM
by measuring the viability of MDA-MB-231 cells and wild-type and
egfr
/
MEFs treated with the drug in the
presence of PP2 or SB203580. In all three cell types, SB203580
significantly inhibited AplidinTM action. In contrast, PP2
was effective only in MDA-MB-231 cells, but not in MEFs (Fig.
4C). In agreement with the differential activation of p38
MAPK/JNK, these experiments also confirmed that egfr
/
MEFs are less sensitive to the drug
than wild-type MEFs. Together, these results indicate that the
activation of EGFR, Src, and p38 MAPK/JNK is involved in
AplidinTM action in MDA-MB-231 cells, whereas only EGFR and
p38 MAPK/JNK (but not Src) seem to be involved in the cytotoxic effect
of AplidinTM in MEFs. EGFR activation participates in, but
is not sufficient for, the induction of apoptosis by
AplidinTM.
View larger version (51K):
[in a new window]
Fig. 4.
Involvement of EGFR, Src, and p38 MAPK/JNK in
AplidinTM action. A, EGFR was involved in
AplidinTM-induced apoptosis. Shown are dot-plot diagrams of
Alexa 488-conjugated annexin V/propidium iodide flow cytometry of
egfr /
and wild-type MEFs after treatment
with AplidinTM. Cells were incubated for 5 h in the
absence (Control) or presence of 500 nM
AplidinTM (APL) in serum-free medium. The cells
were labeled with Alexa 488-conjugated annexin V and propidium iodide
and analyzed by flow cytometry. The lower left quadrants of
each panel show the viable cells (Alexa 488
/propidium
iodide
); the upper right quadrants contain the
nonviable, necrotic, and/or late apoptotic cells (Alexa
488+/propidium iodide+); and the lower
right quadrants represent the early apoptotic cells (Alexa
488+/propidium iodide
). The
numbers in each quadrant represent the percentage of cells.
The results are representative of one of two experiments, which gave
similar results. B, upper panels, the levels of
activated p38 MAPK and JNK in egfr
/
and
wild-type MEFs treated with AplidinTM (500 nM)
for the indicated times or left untreated (control (C)) were
measured by Western blotting using phospho (P)-specific
antibodies as described in the legend to Fig. 2. Antibodies against
total p38 MAPK or JNK were used as controls. To reduce basal enzyme
activities, cells were incubated overnight in serum-free medium. Three
independent experiments were performed. Values from a representative
experiment are shown. Lower panels, shown are the effects of
SB203580, PP2, and GSH on p38 MAPK and JNK activation by
AplidinTM. Cells were pretreated with SB203580
(SB; 30 µM), PP2 (25 µM), GSH
(15 mM), or vehicle (control (C)) for 1 h
before AplidinTM addition (500 nM, 1 h) as
indicated. Conditions were as reported for A. C,
shown is the inhibition of AplidinTM cytotoxicity in human
MDA-MB-231 cells and wild-type and egfr
/
MEFs by SB203580 (30 µM) or PP2 (25 µM).
Cultures were pretreated with the inhibitors for 2 h before
AplidinTM (500 nM) addition, and the total cell
mass in the cultures was measured 16 h (MDA-MB-231 and
egfr
/
fibroblasts) or 4 h (wild-type
fibroblasts) later by the crystal violet staining method. Means ± S.D. of values obtained in AplidinTM-untreated controls are
shown. ***, p < 0.001.
) (40), an effect that, in the case of
H2O2, requires Src-dependent EGFR transactivation (31). These data led us to investigate the putative role of reactive oxygen species in AplidinTM action. As
shown in Fig. 5 (A and
B) (data not shown), pretreatment of MDA-MB-231 cells with
various antioxidants such as ascorbic acid, DPI (an inhibitor of
flavin-containing enzymes such as NAD(P)H oxidase), Tiron (a scavenger
of superoxide ions), and vitamin E or addition of catalase
(H2O2-degrading enzyme) did not affect the
induction of EGFR phosphorylation by AplidinTM. In
contrast, two compounds that raise the cellular level of GSH,
N-acetylcysteine (a GSH precursor) and ebselen (a GSH
peroxidase mimetic), reduced EGFR phosphorylation by
AplidinTM (Fig. 5C). Confirming the involvement
of GSH in AplidinTM action, treatment of cells with
exogenous GSH diminished, whereas BSO (a specific inhibitor of
-glutamylcysteine synthetase) increased, AplidinTM-induced EGFR phosphorylation (Fig.
5C). None of the agents tested (N-acetylcysteine,
ebselen, DPI, and Tiron) modified EGFR activation by its EGF ligand
(Fig. 5D).
View larger version (32K):
[in a new window]
Fig. 5.
AplidinTM-induced EGFR
phosphorylation relies on cellular GSH depletion. Phosphorylated
(P) and total EGFRs were measured by immunoprecipitation and
Western blotting as described in the legend to Fig. 3. A,
some antioxidants (N-acetylcysteine (NAC), 10 mM; and ebselen (EBS), 40 µM), but
not others (DPI, 10 µM; and Tiron (Tir), 10 mM), inhibit AplidinTM-induced EGFR
phosphorylation. Where indicated, MDA-MB-231 cells were pretreated with
the antioxidants 1 h before addition of AplidinTM, and
extracts were prepared 1 h later. B, exogenous catalase
does not affect EGFR phosphorylation by AplidinTM. Cells
were incubated with catalase (Cat; 1000 units/ml) for 1 h as indicated before AplidinTM (APL) treatment.
As a control, we used cells treated with H2O2
(10 mM) plus or minus catalase pretreatment. C,
cellular GSH content modulates EGFR phosphorylation by
AplidinTM. Treatment of cells with exogenous GSH (15 mM, 1 h) or BSO (1 mM, 24 h) before
AplidinTM had opposite effects on the induction of EGFR
phosphorylation. In A-C, AplidinTM was used at
500 nM for 1 h. D, antioxidants do not
affect EGF-induced EGFR phosphorylation. Cells were pretreated with
DPI, Tiron, N-acetylcysteine, or ebselen for 1 h at the
doses indicated for A before addition of EGF (20 ng/ml, 10 min). Results shown correspond to a representative experiment. The
experiments shown in A and C were performed three
times, and those in B and D were performed twice.
C, control.
View larger version (50K):
[in a new window]
Fig. 6.
Sustained activation of p38 MAPK and JNK by
AplidinTM is partially dependent on GSH
depletion and Src and EGFR activation. A, intracellular
GSH levels modulate the activation of p38 MAPK and JNK by
AplidinTM. MDA-MB-231 cells were treated with the indicated
agents that modulate GSH levels at the doses indicated in the legend to
Fig. 4 for 1 h (GSH and N-acetylcysteine
(NAC)) or 24 h (BSO) and thereafter were treated with
AplidinTM (500 nM) or left untreated (control
(C)). Levels of activated p38 MAPK or JNK were measured by
Western blotting using phospho (P)-specific antibodies as
described in the legend to Fig. 2. Antibodies against total p38 MAPK or
JNK were used as controls. Three independent experiments were done.
Values from a representative experiment are shown. B, Src
activation by AplidinTM depends on GSH depletion. Cells
were treated with exogenous GSH (15 mM, 1 h) or BSO (1 mM, 24 h) and thereafter treated with
AplidinTM (500 nM). Activated Src was measured
45 min later by Western blotting using anti-phospho-Tyr418
antibody. Antibody against total Src was used as a control.
C, inhibition of Src and EGFR reduces p38 MAPK and JNK
activation by AplidinTM. Cells were pretreated with PP2 (25 µM) or AG1478 (20 µM) before addition of
AplidinTM (500 nM). Phospho-p38 MAPK and
phospho-JNK were measured as described for A in extracts
prepared 1 h after AplidinTM treatment. The experiment
was performed three times. Values from a representative experiment are
shown. D, AplidinTM has a dominant effect over
EGF on p38 MAPK and JNK activity. Cells were treated with EGF (20 ng/ml), AplidinTM (500 nM), or both for 1 h, and the levels of phospho-p38 MAPK and phospho-JNK were measured as
described for A. Two independent experiments gave the same
results.
View larger version (54K):
[in a new window]
Fig. 7.
Human renal cancer cells are sensitive to
AplidinTM. A, phase-contrast micrographs of
A-498 and ACHN cell cultures treated with vehicle (Control,
C) or AplidinTM for 24 h in normal growth
medium. Scale bar = 40 µM. B,
time course of the induction of EGFR phosphorylation by
AplidinTM in A-498 cells. Total and phosphorylated
(P) EGFRs were measured by immunoprecipitation and Western
blotting as described in the legend to Fig. 3. C, effect of
AplidinTM on Src activity in A-498 (upper
panels) and ACHN (lower panels) cells. Total and
activated Src proteins were measured by Western blotting using anti-Src
antibody and the activation-linked anti-phospho-Tyr418
antibody, respectively, as described under "Experimental
Procedures." D, activation of p38 MAPK (upper
panels) and JNK (lower panels) by AplidinTM
in A-498 (left panels) and ACHN (right panels)
cells. The inhibitory effect of SB203580 (30 µM) is
shown. Total and activated forms of both kinases were measured by
Western blotting using appropriate antibodies as described in the
legend to Fig. 2. In all experiments, AplidinTM was used at
500 nM. Results shown correspond to a representative
experiment. The experiments shown in A-C were performed
three times, and those in D were performed twice.
Role of EGFR, Src, and p38 MAPK/JNK in
AplidinTM-induced apoptosis in renal ACHN and A-498 cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, multiple physiological and nonphysiological stimuli and
agents transactivate EGFR, including redox stress, activation of
various G-protein-coupled receptors and voltage-gated Ca2+
channels, cytokine receptors, osmotic stress, and UV and
-radiation (41, 42). The intensity and duration of EGFR transactivation, in
addition to substrate availability and other signals, are critical for
the cellular response, either proliferation or alternative pathways.
Remarkably, AplidinTM has a dominant effect on EGF,
altering the binding of adaptor proteins and inducing stronger
activation of JNK and p38 MAPK and apoptosis also in EGF-treated
MDA-MB-231 cells. Moreover, these cells harbor mutated p53 and
ras genes and high levels of activated ERK and Akt, two
enzymes that promote cell survival and proliferation. Together, these
data emphasize the potent cytotoxic activity of
AplidinTM.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. E. Wagner for providing the
wild-type and egfr/
MEFs, Dr. J. Martín for help with the Src experiments and Dr. J. Brugge for
anti-Src antibody. We are also grateful to Robin Rycroft for valuable
assistance in the preparation of the English manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant SAF2001-2291 from the Plan Nacional de Investigación y Desarrollo of Spain.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.
¶ Both authors contributed equally to this work.
Recipient of a postdoctoral fellowship from the Consejo
Superior de Investigaciones Científicas.
** Recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura of Spain.
To whom correspondence should be addressed: Inst. de
Investigaciones Biomédicas "Alberto Sols," Arturo Duperier,
4, E-28029 Madrid, Spain. Tel.: 34-91-585-4640; Fax: 34-91-585-4587;
E-mail: amunoz@iib.uam.es.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M201010200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MEFs, mouse embryo fibroblasts; DPI, diphenyleneiodonium; BSO, L-buthionine (SR)-sulfoximine; PDGFR, platelet-derived growth factor receptor; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sakai, R., Rinehart, K. L., Kishore, V., Kundu, B., Faircloth, G., Gloer, J. B., Carney, J. R., Namikoshi, M., Sun, F., Hughes, R. G., Jr., García-Grávalos, D., García deq Uesada, T., Wilson, G. R., and Heid, R. M. (1996) J. Med. Chem. 39, 2819-2834[CrossRef][Medline] [Order article via Infotrieve] |
2. | Lobo, C., García-Pozo, S. G., Núñez de Castro, I., and Alonso, F. J. (1997) Anticancer Res. 17, 333-336[Medline] [Order article via Infotrieve] |
3. | Depenbrock, H., Peter, R., Faircloth, G., Manzanares, I., Jimeno, J., and Hanauske, A. R. (1998) Br. J. Cancer 78, 739-744[Medline] [Order article via Infotrieve] |
4. | Faircloth, G., Hanauske, A., Depenbrock, H., Peter, R., Crews, C., Manzanares, I., Meely, K., Grant, W., and Jimeno, J. (1997) in Proceedings of the 88th Annual Meeting of the American Association for Cancer Research, April 12-16, 1997, Vol. 38, p. 103, San Diego, CA, Abstr. 692 |
5. | Raymond, E., Paz-Ares, L., Izquierdo, M., Belanger, K., Maroun, L., Bowman, A., Anthoney, A., Jodrell, D., Armand, J. P., Cortes-Funes, H., Germa-Lluch, J., Twelves, C., Celli, C., Guzman, C., and Jimeno, J. in Proceedings of the 11th European Cancer Conference, October 21-25, 2001, p. S32, Abstr. 197, Lisbon, Portugal |
6. | Urdiales, J. L., Morata, P., Núñez de Castro, I., and Sánchez-Jiménez, F. (1996) Cancer Lett. 102, 31-37[CrossRef][Medline] [Order article via Infotrieve] |
7. | Gómez-Fabre, P. M., De, Pedro, E., Medina, M. A., Núñez de Castro, I., and Márquez, J. (1997) Cancer Lett. 113, 141-144[CrossRef][Medline] [Order article via Infotrieve] |
8. | Broggini, M., Marchini, S., D'Incalci, M., Taraboletti, G., Giavazzi, R., Faircloth, G., and Jimeno, J. in Proceedings of the 11th National Cancer Institute-European Organisation for Research and Treatment of Cancer-American Association for Cancer Research Symposium on New Drugs in Cancer Therapy, November 7-10, 2000, p. 86, Abstr. 214, Amsterdam |
9. | Lowe, S. W., Ruley, H. E., Jacks, T., and Housman, D. E. (1993) Cell 74, 957-967[Medline] [Order article via Infotrieve] |
10. | Fisher, D. E. (1994) Cell 78, 539-542[Medline] [Order article via Infotrieve] |
11. | Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve] |
12. |
Chen, Y.-R.,
Wang, X.,
Templeton, D.,
Davis, R. J.,
and Tan, T.-H.
(1996)
J. Biol. Chem.
271,
31929-31936 |
13. | Yu, R., Shtil, A., Tan, T.-H., Roninson, I. B., and Kong, A.-N. T. (1996) Cancer Lett. 107, 73-81[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Seimiya, H.,
Mashima, T.,
Toho, M.,
and Tsuruo, T.
(1997)
J. Biol. Chem.
272,
4631-4636 |
15. | Shtil, A., Mandlekar, S., Yu, R., Walter, R. J., Hagen, K., Tan, T.-H., Roninson, I. B., and Kong, A.-N. T. (1998) Oncogene 18, 377-384 |
16. | Sánchez-Pérez, I., and Perona, R. (1999) FEBS Lett. 453, 151-158[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Srivastava, R. K., Mi, Q.-S.,
Hardwick, J. M.,
and Longo, D. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3775-3780 |
18. |
Fan, M.,
Goodwin, M. E.,
Birrer, M. J.,
and Chambers, T. C.
(2001)
Cancer Res.
61,
4450-4458 |
19. |
Yujiri, T.,
Sather, S.,
Fanger, R. S.,
and Johnson, G. L.
(1998)
Science
282,
1911-1914 |
20. |
Wisdom, R.,
Johnson, R. S.,
and Moore, C.
(1999)
EMBO J.
18,
188-197 |
21. |
Hayakawa, J.,
Ohmichi, M.,
Kurachi, H.,
Ikegami, H.,
Kimura, A.,
Matsuoka, T.,
Jikihara, H.,
Mercola, D.,
and Murata, Y.
(1999)
J. Biol. Chem.
274,
31648-31654 |
22. | Davis, R. J. (2000) Cell 103, 239-252[Medline] [Order article via Infotrieve] |
23. |
Chen, Y.-R.,
Wang, W.,
Kong, A.-N. T.,
and Tan, T.-H.
(1998)
J. Biol. Chem.
273,
1769-1775 |
24. | Herr, I., Wilhem, D., Bohler, T., Angel, P., and Debatin, M. (1999) Int. J. Cancer 80, 417-424[CrossRef][Medline] [Order article via Infotrieve] |
25. | Sambrook, J., Fritsch, E. F., and Maniatis, T. E. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
26. | Abella, B., Faircloth, G., López-Lázaro, L., Guzmán, C., and Jimeno, J. (2002) Eur. J. Cancer 38, 1395-1404[CrossRef][Medline] [Order article via Infotrieve] |
27. | Gilhooly, E. M., and Rose, D. P. (1999) Int. J. Oncol. 15, 267-270[Medline] [Order article via Infotrieve] |
28. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
29. | Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233[CrossRef][Medline] [Order article via Infotrieve] |
30. | Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Chen, K.,
Vita, J. A.,
Berk, B. C.,
and Keaney, J. F., Jr.
(2001)
J. Biol. Chem.
276,
16045-16050 |
32. | Levitzki, A., and Gazit, A. (1995) Science 267, 1782-1788[Medline] [Order article via Infotrieve] |
33. |
Yoshizumi, M.,
Abe, J.,
Haendeler, J.,
Huang, Q.,
and Berk, B. C.
(2000)
J. Biol. Chem.
275,
11706-11712 |
34. | Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485[Medline] [Order article via Infotrieve] |
35. | Margolis, B., Li, N., Koch, A., Mohammadi, M., Hurwitz, D. R., Zilberstein, A., Ullrich, A., Pawson, T., and Schlessinger, J. (1990) EMBO J. 9, 4374-4380 |
36. |
Walton, G. M.,
Chen, W. S.,
Rosenfeld, M. G.,
and Gill, G. N.
(1990)
J. Biol. Chem.
265,
1750-1754 |
37. |
Biscardi, J. S.,
Maa, M.-C.,
Tice, D. A.,
Cox, M. E.,
Leu, T.-H.,
and Parsons, S. J.
(1999)
J. Biol. Chem.
274,
8335-8343 |
38. |
Andreev, J.,
Galisteo, M. L.,
Kranenburg, O.,
Logan, S. K.,
Chiu, E. S.,
Okigaki, M.,
Cary, L. A.,
Moolenaar, W. H.,
and Schlessinger, J.
(2001)
J. Biol. Chem.
276,
20130-20135 |
39. | Pascal, S. M., Singer, A. U., Gish, G., Yamazaki, T., Shoelson, S. E., Pawson, T., Kay, L. E., and Forman, K.-J. D. (1994) Cell 77, 461-472[Medline] [Order article via Infotrieve] |
40. |
Lo, Y. Y. C.,
Wong, J. M. S.,
and Cruz, T. F.
(1996)
J. Biol. Chem.
271,
15703-15707 |
41. | Zwick, E., Hackel, P. O., Prenzel, N., and Ullrich, A. (1999) Trends Pharmacol. Sci. 20, 408-412[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Kamata, H.,
Shibukawa, Y.,
Oka, S.-I.,
and Hirata, H.
(2000)
Eur. J. Biochem.
267,
1933-1944 |
43. | Maa, M.-C., Leu, T.-H., McCarley, D. J., Schatzman, R. C., and Parsons, S. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6981-6985[Abstract] |
44. | Chang, J. H., Wilson, L. K., Moyers, J. S., Zhang, K., and Parson, S. J. (1993) Oncogene 8, 959-967[Medline] [Order article via Infotrieve] |
45. |
Stover, D. R.,
Becker, M.,
Liebetanz, J.,
and Lydon, N. B.
(1995)
J. Biol. Chem.
270,
15591-15597 |
46. | Osherov, N., and Levitzki, A. (1994) Eur. J. Biochem. 225, 1047-1053[Abstract] |
47. |
Lavoie, J. N.,
Champagne, C.,
Gingras, M.-C.,
and Robert, A.
(2000)
J. Cell. Biol.
150,
1037-1056 |
48. | Pelengaris, S., Rudolph, B., and Littlewood, T. (2000) Curr. Opin. Genet. Dev. 10, 100-105[CrossRef][Medline] [Order article via Infotrieve] |
49. | De Vries, N., and De Flora, S. (1993) J. Cell. Biochem. Suppl. 17F, 270-277 |
50. |
Guyton, K. Z.,
Liu, Y.,
Gorospe, M., Xu, Q.,
and Hölbrook, N. J.
(1996)
J. Biol. Chem.
271,
4138-4142 |
51. |
Wilmer, W. A.,
Tan, L. C.,
Dickerson, J. A.,
Danne, M.,
and Rovin, B. H.
(1997)
J. Biol. Chem.
272,
10877-10881 |
52. | Liu, Y., Guyton, K. Z., Gorospe, M., Xu, Q., Lee, J. C., and Hölbrook, N. J. (1996) Free Radic. Biol. Med. 21, 771-781[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Liu, B.,
Andrieu-Abadie, N.,
Levade, T.,
Zhang, P.,
Obeid, L. M.,
and Hannun, Y. A.
(1998)
J. Biol. Chem.
273,
11313-11320 |
54. | Fernandes, R. S., and Cotter, T. G. (1994) Biochem. Pharmacol. 48, 675-681[CrossRef][Medline] [Order article via Infotrieve] |
55. | Palmer, H. J., and Paulson, K. E. (1997) Nutr. Rev. 55, 353-361[Medline] [Order article via Infotrieve] |
56. | Wilhelm, D., Bender, K., Knebel, A., and Angel, P. (1999) Mol. Cell. Biol. 17, 4792-4800[Abstract] |
57. | Chen, Z., Seimiya, M., Naito, M., Mashima, T., Kizaki, A., Dan, S., Imaizumi, M., Ichijo, H., Miyazono, K., and Tsuruo, T. (1999) Oncogene 18, 173-180[CrossRef][Medline] [Order article via Infotrieve] |
58. | Shaulian, E., and Karin, M. (2001) Oncogene 20, 2390-2400[CrossRef][Medline] [Order article via Infotrieve] |
59. | Bacus, S. S., Gudkov, A. V., Lowe, M., Lyass, L. K., Yung, Y., Komarov, A. P., Keyomarsi, K., Yarden, Y., and Seger, R. (2001) Oncogene 20, 147-155[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Mandlekar, S., Yu, R.,
Tan, T.-H.,
and Kong, A.-N. T.
(2000)
Cancer Res.
60,
5995-6000 |
61. |
Einchhorst, S. T.,
Müller, M., Li-,
Weber, M.,
Schulze-Bergkamen, H.,
Angel, P.,
and Krammer, P. H.
(2000)
Mol. Cell. Biol.
20,
7826-7837 |