Inhibition of NF-
B Sensitizes A431 Cells to Epidermal Growth Factor-induced Apoptosis, whereas Its Activation by Ectopic Expression of RelA Confers Resistance*
Ruby John Anto,
Manickam Venkatraman
and
Devarajan Karunagaran
From the
Division of Cancer Biology, Rajiv Gandhi Center for Biotechnology,
Thiruvananthapuram, Kerala-695014, India
Received for publication, February 19, 2003
, and in revised form, April 16, 2003.
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ABSTRACT
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Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically
induces apoptosis in cells that overexpress its receptor. We demonstrate for
the first time that the EGF-induced apoptosis is accelerated if NF-
B is
inactivated. To inactivate NF-
B, human epidermoid carcinoma cells
(A431) that overexpress EGF receptor were stably transfected with an
I
B-
double mutant construct. Under the NF-
B-inactivated
condition, A431 cells were more sensitive to EGF with decreased cell viability
and increased externalization of phosphatidylserine on the cell surface, DNA
fragmentation, and activation of caspases (3 and 8 but not 9), typical
features of apoptosis. These results were further supported by the
potentiation of the growth inhibitory effects of EGF by chemical inhibitors of
NF-
B (curcumin and sodium salicylate) and the protective role of RelA
evidenced by the resistance of A431-RelA cells (stably transfected with RelA)
to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431
cells induced DNA binding activity of NF-
B (p50 and RelA) and the
expression of c-IAP1, a downstream target of NF-
B. A431-RelA cells
exhibited spontaneous phosphorylation of Akt (a downstream target of
phosphatidylinositol 3-kinase and regulator of NF-
B) and EGF treatment
stimulated it further. Blocking this basal Akt phosphorylation with LY294002,
an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability
but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise
resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor
an anti-apoptotic role for NF-
B in the regulation of EGF-induced
apoptosis.
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INTRODUCTION
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Epidermal growth factor
(EGF)1 is a
polypeptide (6-kDa) that belongs to the EGF family of ligands (heparin binding
EGF, transforming growth factor-
, amphiregulin,
-cellulin,
epiregulin, and neuregulins) binding to specific cell surface receptors
(1,
2). Upon ligand binding, the
epidermal growth factor receptor (EGFR) dimerizes, autophosphorylates itself,
and recruits a cascade of signaling molecules before transmitting potent
mitogenic signals in many cellular systems
(1,
3). EGFR is overexpressed in a
number of human malignancies including cancers of the lung, head and neck,
brain, bladder, and breast (4).
Furthermore, increased EGFR expression correlates with a poorer clinical
outcome for patients with breast and ovarian cancers
(5,
6). Whereas EGF is a potent
mitogen, it paradoxically induces apoptosis in cells that overexpress EGFR
such as A431 (7).
Experimentally increasing the level of EGFR expression in epithelial,
mesenchymal, or glial cells also leads to ligand-dependent apoptosis
(8). Another ligand of the EGF
family, heregulin (also known as neuregulin), is known to induce apoptosis in
cells that overexpress ErbB2, the second member of the EGFR family
(9). In addition, epiregulin
also inhibited cell growth in EGFR-overexpressing cells
(10). Induction of
amphiregulin mRNA was observed in EGF-induced apoptosis
(11) and interaction of EGF
with pro-heparin-binding EGF leads to growth inhibition and apoptosis
(12). Growth factors other
than EGF such as platelet-derived growth factor and hepatocyte growth factor
can also trigger cell cycle arrest and death in a variety of cells
(13,
14) and in addition, EGF
enhanced the apoptotic effect of platelet-derived growth factor
(14). An EGF-related protein,
Cripto-1, promotes apoptosis in HC-11 mouse mammary epithelial cells
(15). Anoikis, activation of
EGFR tyrosine kinase, Ras-MAP kinase signaling, and the elevation of Stat1 and
p21 levels have been advocated as mechanisms driving EGF-induced apoptosis
(11,
1618)
but, the actual mechanism appears to be more elusive and complicated.
Apoptosis or programmed cell death is a physiological process characterized
by distinct morphological and biochemical features that include membrane
blebbing, chromatin condensation, cytoplasmic shrinking, DNA fragmentation,
and activation of different caspases
(19). Typically two different
pathways, extrinsic receptor-mediated and intrinsic mitochondria-mediated,
leading to apoptosis have been identified
(20,
21). Mostly cytokines of the
tumor necrosis factor (TNF) superfamily induce apoptosis by interaction of the
ligand with its death receptor, which sequentially recruits TNF
receptor-associated death domain, Fas-associated death domain, caspase 8, and
caspase 3, the last then cleaves various substrates leading to apoptosis. In
contrast, the mitochondria-mediated pathway involves the release of cytochrome
c from the mitochondria, and cytochrome c together with
Apaf1 activates caspase 9, and the latter then activates caspase 3, resulting
in apoptosis (20,
21). Tumor cells often evade
apoptosis by expressing several anti-apoptotic proteins such as Bcl-2,
down-regulation and mutation of pro-apoptotic genes and alterations of p53,
PI-3K/Akt, or NF-
B pathways that give them survival advantage and
thereby resist therapy-induced apoptosis
(20).
NF-
B is a family of transcription factors activated by a diverse
number of stimuli including EGF, cytokines, such as TNF-
and
interleukin-1, UV irradiation, and lipopolysaccharides
(22). EGF has been reported to
activate NF-
B in smooth muscle cells, fibroblasts, and in several
EGFR-overexpressing cell lines
(2325).
Binding of I
B to NF-
B masks nuclear localization signals and
prevents its translocation to the nucleus
(26). Stimulation of cells
with a diverse array of stimuli results in phosphorylation of
I
B-
on serines 32 and 36 at its NH2-terminal. This
leads to the ubiquitination and degradation of I
B-
, allowing
NF-
B to translocate to the nucleus and activate transcription
(22,
26). Inhibition of NF-
B
activity potentiates cell killing of human breast cancer and fibrosarcoma cell
lines by TNF-
, ionizing radiation, and daunorubicin
(2729).
NF-
B inhibition sensitized tumors in mice to chemotherapeutic compound
CPT-11-mediated cell killing
(30). NF-
B directly
causes increased expression of proteins that contribute to the survival of
tumor cells such as inhibitors of apoptotic proteins (IAPs)
(31,
32). Results from our
laboratory have shown earlier that ectopic expression of the RelA subunit of
NF-
B into murine fibrosarcoma cells protects them from curcumin-induced
apoptosis (33).
To understand whether NF-
B plays any role in EGF-induced apoptosis,
we used A431 cells that overexpress EGFR and stably transfected them with a
mutant I
B (known to inactivate NF-
B) or RelA (known to activate
NF-
B). Using several parameters to assess apoptosis such as viability,
externalization of phosphatidylserine (PS) on the cell surface, DNA
fragmentation, and activation of caspases we report that A431 cells are more
sensitive to EGF-induced apoptosis under NF-
B-inactivated conditions
whereas its activation confers resistance.
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EXPERIMENTAL PROCEDURES
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Reagents, Chemicals, and AntibodiesEGF (isolated from male
mouse submaxillary glands), Dulbecco's minimum essential medium, and fetal
bovine serum were procured from Invitrogen. Curcumin, sodium salicylate, MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate mixture, and a
mouse monoclonal antibody to
-actin (A-5441) were purchased from Sigma.
Fluorimetric substrates for caspase 3 (Ac-DEVD-AFC number 264157) and caspase
9 (Ac-LEHD-AFC number 218765) were obtained from Calbiochem. Rabbit polyclonal
antibodies to p50 (sc-7178), RelA (sc-109), hemagglutinin (HA) (sc-7392),
I
B-
(sc-271), and c-IAP1 (sc-7943) were procured from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to caspase 8 (1C12
[PDB]
),
rabbit polyclonal PARP antibody (number 9542), and phospho-Akt pathway sampler
kit (number 9916 containing antibodies to Akt and phospho-Akt, and LY294002)
were purchased from Cell Signaling Technology (Beverly, MA), and the mouse
monoclonal EGFR antibody (clone 111) raised against the extracellular domain
of EGFR was a gift from Dr. Yosef Yarden, Weizmann Institute of Science,
Israel.
Cell Lines and CultureHuman epidermoid carcinoma cell line
A431 was obtained from the National Center for Cell Science, Pune, India. The
cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and antibiotics (Invitrogen). Cells
were incubated at 37 °C in a humidified atmosphere of 5% CO2
and 95% air.
Transient and Stable TransfectionsA431-I
B-
cells were transiently transfected with relA in pMT2T vector
(33,
34) using the
calcium-phosphate transfection kit (Invitrogen) according to the
manufacturer's protocol. Stable transfections in A431 cells with relA
in pMT2T vector (co-transfected with pcDNA3) or the empty vector pcDNA3 or
pcDNA3-I
B-
were carried out by the LipofectAMINE method
(35). For the preparation of
liposome solution, 20 µmol/ml of stock (prepared by mixing 6.6 µmol of
dimethyl dioctadecyl ammonium bromide and 13.4 µmol of
dioleyl-L-phosphatidylethanolamine in 1 ml of ethanol) was diluted
into 1 nmol/µl in water. For transfection, the cells were seeded to attain
70% confluence in 35-mm Petri dishes. For each dish, 2 µg of DNA and 24
µl of liposome solution were mixed in 500 µl of Dulbecco's minimum
essential medium free from serum and antibiotics, vortexed, and incubated at
room temperature for 30 min. The liposome solution (1 ml) was layered over the
cells previously rinsed with serum-free medium and left for 4 h in a
CO2 incubator and then the medium was replenished with 20% fetal
bovine serum and reverted back to 10% fetal bovine serum after 24 h. After 72
h, cells were grown in selection medium (400 µg/ml G418) and clones formed
were picked up and maintained separately with 100 µg/ml G418.
Western BlottingCells were lysed in whole cell lysis buffer
(20 mM Tris, pH 7.4, 250 mM NaCl, 2 mM EDTA,
0.1% Triton X 100, 1 mM DTT, 5 µg/ml aprotinin, 5 µg/ml
leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 4
mM sodium orthovanadate). Equal amounts of total protein for each
sample were separated by SDS-PAGE and electrotransferred onto nitrocellulose
filters, probed with the primary antibodies and appropriate
peroxidase-conjugated secondary antibodies, and visualized with the enhanced
chemiluminescence (ECL) method as per the manufacturer's protocol (Amersham
Biosciences). For some experiments, alkaline phosphatase-conjugated secondary
antibodies from Sigma were used and the bands were detected using nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.
Electrophoretic Mobility Shift Assay (EMSA)Cells were
washed with cold PBS and suspended in 150 µl of lysis buffer (10
mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and
0.5 mg/ml benzamidine). The cells were allowed to swell for 30 min, after
which 4.5 µl of 10% Nonidet P-40 was added, vortexed, and centrifuged, and
the pellet was suspended in 25 µl of nuclear extraction buffer (20
mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1
mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 mg/ml
benzamidine). The nuclear extract (8 µg of protein) collected after 30 min
by centrifugation was used to perform EMSA by incubating it with 16 fmol of
32P-end labeled 45-mer double stranded NF-
B oligonucleotide
from the human immunodeficiency virus-1 long terminal repeat
(5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3') in the
presence of 1 µg/ml poly(dI-dC) in a binding buffer (25 mM HEPES
(pH 7.9), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM
DTT, 1% Nonidet P-40, and 5% glycerol) for 30 min at 37 °C and the
DNA-protein complex was resolved using a 6.6% native polyacrylamide gel. The
gels were dried and the radioactive bands were visualized by phosphorimaging
(Bio-Rad Personal FX).
MTT AssayFor MTT assay, 25 µl of MTT solution (5 mg/ml
in PBS) was added to cells (untreated and treated) cultured in 96-well plates.
Cells were incubated for 2 h and 0.1 ml of the extraction buffer (20% sodium
dodecyl sulfate in 50% dimethyl formamide) was added after removal of MTT with
a PBS wash. After an overnight incubation at 37 °C, the optical densities
at 570 nm were measured using a plate reader (Bio-Rad), with the extraction
buffer as a blank. The relative cell viability in percentage was calculated as
(A570 of treated samples/A570 of
untreated samples) x 100.
[3H]Thymidine IncorporationCells grown in
96-well plates were treated with or without the indicated concentrations of
EGF and at the end of 18 h, [3H]thymidine was added to each well
(0.5 µCi/well) and the incubation was continued for a total period of 24 h.
The culture medium was then removed, washed twice with PBS, and the proteins
were precipitated with 5% trichloroacetic acid. The supernatant was removed
and after washing with ethanol, the cells were solubilized with 0.2
N NaOH, and the radioactivity was counted using a liquid
scintillation counter.
Annexin-PI StainingThe cells (104 cells/well)
were seeded in 48-well plates and treated with or without EGF for 16 h. Then
the cells were washed with PBS and treated with 1x assay buffer,
annexin-fluorescein isothiocyanate and propidium iodide (PI) as per the
protocol described in the annexin V apoptosis detection kit (sc-4252 AK) from
Santa Cruz Biotechnology. After 10 20 min, the wells were washed with
PBS and greenish apoptotic cells were viewed using a Nikon fluorescent
microscope and photographed.
Comet AssayComet assay was carried out essentially as
described (36). Briefly, the
cells (treated with or without EGF) were pelleted and resuspended in 0.5% low
melting point agarose at 37 °C and layered on a frosted microscope slide
previously coated with a thin layer of 0.5% normal melting agarose and kept
for 5 min at 4 °C. After solidification, the slides were immersed in
lysing solution (2.5 M NaCl, 100 mM EDTA, 10
mM Tris, pH 10.5, 1% Triton X-100, and 10% Me2SO) for 1
h at 4 °C. The slides were then electrophoresed for 20 to 30 min at 25 V.
The slides after electrophoresis were washed with 0.4 M Tris (pH
7.5) and stained with ethidium bromide (1 µg/ml) and observed under a Nikon
fluorescent microscope.
Assays of Caspase 3 and Caspase 9 The enzymatic activities
of caspase 3 or caspase 9 were assayed spectrofluorimetrically
(37). Briefly, the whole cell
lysate was incubated with 50 µM fluorimetric substrates of
caspase 3 (Ac-DEVD-AFC) or caspase 9 (Ac-LEHD-AFC) in a total volume of 500
µl of reaction buffer (50 mM HEPES-KOH, pH 7.0, 10% glycerol,
0.1% CHAPS, 2 mM EDTA, 2 mM DTT) at 37 °C for 1 h.
The released AFC was quantitated using a spectrofluorimeter (PerkinElmer LS-50
B) with the excitation and emission wavelengths of 380 and 460 nm,
respectively. Values of relative fluorescence units released per mg of protein
were calculated.
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RESULTS
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EGF Induces NF-
B DNA Binding Activity in A431-Neo but
Not A431-I
B-
CellsThe human
epidermoid carcinoma cell line, A431, overexpresses EGFRs (about 2 x
106 EGFRs/cell) and has been extensively used as a model system to
study the effects of EGF on cell proliferation
(7,
3840).
To study the role of NF-
B in EGF-induced apoptosis, we used A431 cells
and stably transfected them with either the empty vector (pcDNA3) or
pcDNA3-I
B-
, a double mutant construct in which both the serines
(32 and 36) at the amino-terminal of I
B-
are mutated to alanine.
Because the double mutant form of I
B-
lacks the crucial serine
residues that need to be phosphorylated by NF-
B activators, it is
popularly employed to strongly inhibit NF-
B
(28). As the construct,
pcDNA3-I
B-
, contains the hemagglutinin tag (HA tag), Western
blotting of HA protein was used to ascertain the presence of the mutant
I
B protein. As expected, all six clones of A431-I
B-
cells
(selected by G418) showed the presence of HA protein whereas the A431-Neo
cells transfected with control vector (pcDNA3) did not show its expression
(Fig. 1A). Further
experiments with I
B-transfected cells were done using only clone 6 of
A431-I
B-
cells (showing very high HA expression) unless stated
otherwise. Western blotting with a polyclonal I
B-
antibody
confirmed that clone 6 had indeed a higher level of I
B-
expression compared with that in the parental as well as A431-Neo cells
(Fig. 1B). For the
Western blots,
-actin was used as a control and the results confirm
equal loading of the samples (Fig. 1,
A and B). To see whether transfection procedures
affected the level of EGFR, Western blotting was carried out in parental,
A431-Neo, and A431-I
B-
cells and the results confirmed that EGFR
levels remained unaffected in these cells
(Fig. 1C). Because the
very purpose of stable transfection was to inactivate NF-
B, it was
logical to check whether EGF could stimulate the NF-
B DNA binding
activity in A431-Neo and A431-I
B-
cells (clone 6) by EMSA.
Whereas 50 ng/ml EGF enhanced the NF-
B DNA binding activity in A431-Neo
cells, even 100 ng/ml EGF could not induce it in A431-I
B-
cells
and in the absence of EGF there were no active DNA-binding complexes of
NF-
B in both the cells (Fig.
1D). To confirm whether the active complex contains the
classical NF-
B partners, p50 and RelA, the nuclear extracts prepared
from A431-Neo cells stimulated with 50 ng/ml EGF were incubated with
antibodies to RelA or p50 and then EMSA was carried out. As shown in
Fig. 1E, both
antibodies shifted the active NF-
B complex (supershift) whereas
incubation with excess of an unlabeled oligonucleotide containing the
NF-
B binding site removed the active complex.
Fig. 1E also shows
that transient transfection of A431-I
B-
cells with relA
restored the NF-
B active complex, and relA transfection was
used by us earlier to constitutively activate NF-
B in L-929 cells
(33). These results confirm
that the A431-I
B-
cells express the mutant form of
I
B-
that inactivated NF-
B and hence, EGF could not
stimulate NF-
B DNA binding activity in these cells. The results also
indicate that EGF can induce the NF-
B DNA binding activity in A431-Neo
cells and the active NF-
B complex contains the heterodimers, p50 and
RelA. In addition, the results show that RelA, being one of the heterodimeric
partners of active NF-
B, favors the formation of active NF-
B DNA
binding complexes and has the potential to reverse the
NF-
B-inactivating effect of I
B.

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FIG. 1. Western blotting for HA and I B- , and assessment of
NF- B DNA binding activity in transfected and untransfected A431
cells. A, A431 cells were transfected with pcDNA3 vector or the
pcDNA3-I B- construct using LipofectAMINE and the G418-resistant
clones were selected as described under "Experimental Procedures."
Cell lysates (40 µg of protein) from the vector-transfected A431-Neo cells
and the different clones of A431-I B- cells were subjected to
SDS-PAGE and immunoblotted with HA antibody or -actin (control) by ECL
as described under "Experimental Procedures." B, cell
lysates from A431, A431-Neo, and the clone-6 of A431-I B- cells
were subjected to Western blotting with an antibody to I B- or
-actin (control) as described above. C, similarly, the cell
lysate from A431, A431-Neo, or A431-I B- cells was subjected to
Western blotting with an antibody to EGFR or -actin (control).
D, A431-Neo cells or A431-I B- cells grown in 35-mm
Petri dishes (1 x 106 cells/dish) were treated with EGF at
the indicated concentrations at 37 °C for 1 h. Nuclear extracts were
prepared and EMSA was done as described under "Experimental
Procedures." E, A431-Neo cells were treated with 50 ng/ml EGF
as described above and EMSA was done as before. The nuclear extracts from
EGF-stimulated cells were also incubated with either RelA or p50 antibody or
with 10 times excess of unlabeled oligo. One of the lanes had the nuclear
extract prepared from A431-I B- cells transiently transfected
with relA as described under "Experimental Procedures"
and another lane contained the labeled oligo (free probe) without the addition
of nuclear extract. All the experiments above were done at least two times
with similar results. The arrowheads shown in panels D and
E indicate the positions of the active DNA-binding complexes of
NF- B.
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A431-I
B-
Cells Are More Sensitive to
EGF-induced Cytotoxicity and DNA Fragmentation Than A431-Neo
CellsTo study the effects of EGF on cell growth under conditions
of NF-
B inactivation, A431-Neo and A431-I
B-
cells were
exposed to various concentrations of EGF and the cell viability in percentage
over untreated control was determined after 72 h by MTT assay. The MTT assay
is a convenient screening assay for the measurement of cell death while it
does not discriminate between apoptosis and necrosis. EGF treatment at 0.01
and 0.1 ng/ml had a stimulatory effect on A431-Neo cells with cell viabilities
of 130 and 110%, respectively, if the viability of untreated control was taken
as 100% at the end of 72 h (Fig.
2A). In contrast, EGF inhibited the
A431-I
B-
cells with only 81 and 67% of cells being alive for the
concentrations of 0.01 and 0.1 ng/ml, respectively, compared with the control
(Fig. 2A). As can be
seen in Fig. 2A, EGF
at a concentration of 1 ng/ml and above was inhibitory to both A431-Neo and
A431-I
B-
cells. Thus, even at concentrations that were
stimulatory to A431-Neo cells, EGF inhibited the growth of
A431-I
B-
cells (Fig.
2A) suggesting that A431-I
B-
cells are much
more sensitive to EGF-induced cytotoxicity and these results were also
confirmed in 2 other clones of A431-I
B-
(data not shown). We
also examined the effect of EGF on DNA synthesis by the method of thymidine
incorporation after exposing the cells to different concentrations of EGF
(110 ng/ml) found to be inhibitory to both A431-Neo and
A431-I
B-
cells by the MTT assay. EGF inhibited DNA synthesis in
both A431-Neo and A431-I
B-
cells in a dose-dependent manner and
again the A431-I
B-
cells were more sensitive to EGF-induced
inhibition of DNA synthesis (Fig.
2B). DNA fragmentation is another hallmark of apoptosis
and to detect this, we have used the single cell gel electrophoresis (Comet
assay), a sensitive technique that allows detection of DNA strand breaks. DNA
strand breaks create fragments that when embedded in agarose gels migrate in
an electric field. Cells with damaged DNA when stained with ethidium bromide
appear like a comet and the length of the comet tail represents the extent of
DNA damage. Fig. 2C
clearly indicates that well formed comets are more in number in
A431-I
B-
than A431-Neo cells when induced with 5 ng/ml EGF while
the untreated cells did not exhibit the comet morphology in both the cells.
These results suggest that A431-I
B-
cells with inactivated
NF-
B are more susceptible to cell death induced by EGF compared with
A431-Neo cells.
Relatively More A431-I
B-
Cells Undergo
EGF-induced Externalization of PhosphatidylserineTo assess whether
the cell death induced by EGF involves typical changes encountered during
apoptosis, we first looked for changes in PS on the cell membrane. Under
defined salt and calcium concentrations, annexin V is predisposed to bind PS
that is externalized to the cell surface in the very early stages of apoptosis
(41,
42). Hence, apoptotic cells
were detected using annexin V labeled with fluorescein isothiocyanate and
photographed with a camera-attached fluorescent microscope. Addition of PI
helps to distinguish the early apoptotic cells from late apoptotic or necrotic
cells because PI cannot enter the cells in the early stages of apoptosis when
the membrane integrity is intact
(41,
42). In A431-I
B-
cells 37 and 65% were annexin positive after treatment with 5 and 10 ng/ml
EGF, respectively, whereas only 9 and 23% of the A431-Neo cells showed annexin
positivity (greenish yellow) for the same corresponding EGF concentrations
(Fig. 3). Untreated A431-Neo
and A431-I
B-
cells either did not show annexin positivity or had
a very minimum number of positively stained cells
(Fig. 3). However, a small
percentage of A431-Neo and A431-I
B-
cells also showed typical PI
staining (yellowish red) suggesting the appearance of late apoptotic or
necrotic cells with 10 ng/ml EGF treatment
(Fig. 3). These results
indicate that in comparison with A431-Neo cells, a relatively large number of
A431-I
B-
cells exhibit externalization of PS, a typical feature
of apoptosis upon treatment with EGF.

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FIG. 3. EGF-induced changes in annexin-PI staining. Cells were incubated
with or without the indicated concentrations of EGF for 16 h, and stained for
annexin-PI positivity (not shown in color) as described under
"Experimental Procedures." The same results were confirmed in
another independent experiment.
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A431-I
B-
Cells Are More Sensitive to
EGF-induced Apoptosis That Involves Cleavage of PARP, Activation of Caspases 3
and 8, but Not Caspase 9 In many systems caspase 8 and caspase 9
act as initiator caspases and caspase 3, the effector, signals for the final
execution of the cells. Pro-caspase 8 was cleaved into its active fragments
(p43/41 and p18) by 10 ng/ml EGF in A431-Neo cells while even 5 ng/ml EGF
could easily do it in A431-I
B-
cells as visualized by Western
blotting and the untreated cells did not show any of the cleavage products
(Fig. 4A). Caspase 3
activity was determined spectrofluorimetrically using a substrate,
Ac-DEVD-AFC, an acetylated synthetic tetrapeptide corresponding to the
upstream amino acid sequence of the caspase 3 cleavage site in PARP, and the
fluorphor AFC (7-amino-4-trifluromethyl coumarin). Whereas EGF-induced caspase
3 activity was 2.1-fold more than the untreated control in
A431-I
B-
cells, it was only 1.2-fold more than the control in
A431-Neo cells (Fig.
4B). Caspase 9 activity was also determined
fluorimetrically and EGF treatment could not induce caspase 9 activity in
A431-I
B-
and A431-Neo cells, whereas curcumin used as a positive
control activated caspase 9 in both cells
(Fig. 4C). We also
examined the cleavage of a well characterized caspase 3 substrate, PARP, from
its 116-kDa intact form into the 89-kDa fragment by Western blotting. When
A431-I
B-
cells were treated with 5 or 10 ng/ml EGF, the 116-kDa
form of PARP decreased and the 89-kDa form increased indicating that the
full-length PARP was converted to an apoptotic fragment while the untreated
cells showed only the uncleaved fragment
(Fig. 4D). In A431-Neo
cells, both control and 5 ng/ml EGF-treated cells did not exhibit the cleaved
product and even 10 ng/ml EGF could induce only a slight PARP cleavage
(Fig. 4D). The above
results confirm that the A431-I
B-
cells are more sensitive to
EGF-induced apoptosis than A431-Neo cells by showing increased PARP cleavage
and higher activation of caspases 3 and 8 but not 9 suggesting the operation
of a caspase 8-mediated extrinsic pathway.

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FIG. 4. Effects of EGF on the activities of caspases 8, 3, and 9, and PARP
cleavage. A, cells (1 x 106) were seeded in
35-mm Petri dishes and treated with or without EGF for 24 h. To detect the
active caspase 8 fragments, the cell lysates were resolved on 12% SDS-PAGE,
electrotransferred onto a nitrocellulose membrane, probed with caspase 8
antibody (1:3000), and detected by the alkaline phosphatase method using nitro
blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.
B, cells (1 x 106) were seeded in 35-mm Petri dishes
and treated with or without EGF for 24 h. Fifty micrograms of total protein
was incubated with 50 µM caspase 3 fluorimetric substrate in a
total volume of 500 µl of the reaction buffer and the fluophor released was
quantitated spectrofluorimetrically as described under "Experimental
Procedures." C, cells (1 x 106) were seeded in
35-mm Petri dishes and treated with EGF or curcumin along with untreated
control for 24 h. Fifty micrograms of total protein was incubated with 50
µM caspase 9 fluorimetric substrate and the activity was
quantitated spectrofluorimetrically as above. The reproducibility of these
experiments was ascertained by repeating them at least two times. D,
cells (1 x 106) were seeded in 35-mm Petri dishes and treated
with or without EGF for 24 h. To detect the cleavage of PARP, whole cell
lysate (40 µg) was resolved on a 7.5% polyacrylamide gel,
electrotransferred, probed with PARP antibody (1:3000), and detected by ECL
reagent as described earlier. Similar results were obtained when the
experiment was repeated.
|
|
Chemical Inhibitors of NF-
B Enhance the Susceptibility
of A431-Neo Cells whereas Activation of NF
B by RelA Reverses
the Susceptibility of A431-I
B-
Cells to
EGF-induced CytotoxicityTo know whether chemical inhibitors of
NF-
B would also enhance EGF-induced apoptosis, the A431-Neo cells were
treated with known inhibitors of NF-
B such as sodium salicylate (50
µM) or curcumin (10 µM) for 2 h prior to EGF
treatment. When compared with untreated cells, 77% A431-Neo cells were alive
in wells treated with 5 ng/ml EGF for 24 h
(Fig. 5A). If the
viability of A431-Neo cells treated with 10 µM curcumin alone
for 24 h was taken as 100, treatment with EGF after pretreatment with curcumin
reduced it to 71% (Fig.
5A). Similarly, if 50 µM sodium salicylate
pretreatment is compared with or without EGF the viability of A431-Neo cells
was only 61% (Fig.
5A). If I
B-mediated inhibition of NF-
B can
positively regulate EGF-induced apoptosis, then this effect is expected to be
reversed by NF-
B. As expected, the transient transfection of
relA into A431-I
B-
cells partly reversed the cytotoxic
effect of EGF as measured by an MTT assay
(Fig. 5B).
The higher expression of RelA in the transfected cells was confirmed by
Western blotting and to ensure that proteins were loaded equally,
-actin
controls were used (Fig.
5B, inset). These results suggest that similar
to I
B, NF-
B inhibitors also have the potential to enhance
EGF-induced cell death and NF-
B has a protective role suggested by the
higher level of resistance of A431-I
B-
cells transiently
transfected with relA to EGF-induced cytotoxicity.
RelA Protects A431 Cells from EGF-induced Apoptosis Because
RelA reversed the effect of I
B-
it became relevant to know
whether, on its own, it can protect the parental A431 cells from EGF-induced
apoptosis. To this end, we transfected the A431 cells stably with
relA and confirmed the higher expression of RelA in A431-RelA cells
(clone 1 and clone 2) compared with A431 and A431-Neo cells by Western
blotting (Fig. 6A).
When A431-Neo and A431-RelA cells (clone 1) were compared for their relative
viability in the presence of varying concentrations of EGF by the MTT assay,
A431-RelA cells were notably more resistant to EGF
(Fig. 6B) and these
results were also confirmed using clone 2 (data not shown) but further
experiments were done using clone 1 of A431-RelA cells. Similarly EGF-mediated
inhibition of thymidine incorporation was relatively more in A431-Neo than
A431-RelA cells confirming the protective role of RelA against EGF-mediated
cell death (Fig. 6C).
In addition, varying EGF concentrations even up to 50 ng/ml could not induce
caspase 8 (Fig. 6D) or
caspase 3 (Fig. 6E)
activities or PARP cleavage (Fig.
6F) in A431-RelA cells.

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|
FIG. 6. Western blotting for RelA and EGF-mediated changes in growth, DNA
synthesis, caspase activation, and PARP cleavage. A, A431 cells
were cotransfected with pcDNA3 vector and relA PMT2T construct using
LipofectAMINE and the G418-resistant clones were selected as described under
"Experimental Procedures." Cell lysates (60 µg of protein) from
the vector-transfected A431-Neo cells and different clones of A431-RelA cells
were subjected to SDS-PAGE and immunoblotted with RelA or -actin
(control) antibody by the alkaline phosphatase method as described under
"Experimental Procedures." B, cells were treated with or
without the indicated concentrations of EGF for 24 h and the MTT assay was
done under the conditions described for
Fig. 2. Triplicate samples were
used and the error bars indicate the standard deviations. The results
were confirmed in another independent experiment. C, cells were
treated with or without the indicated concentrations of EGF for 24 h and
thymidine incorporation assays were done under the conditions described for
Fig. 2. Triplicate samples were
used and the error bars indicate the standard deviations. The results
were confirmed in another independent experiment. D, A431-RelA cells
were treated with or without the indicated concentrations of EGF for 24 h and
caspase 8 activity was determined as described for
Fig. 4. E, cells were
treated with or without the indicated concentrations of EGF for 24 h and
caspase 3 activity was determined as described for
Fig. 4. F, cells were
treated with or without the indicated concentrations of EGF for 24 h and PARP
cleavage was determined as described for
Fig. 4.
|
|
EGF Up-regulates the Expression of c-IAP1 in A431-Neo Cells and Its
Basal Expression Is Higher in A431-RelA CellsAs IAP is considered
to be one of the survival proteins induced by NF-
B, it was of interest
to study the effect of EGF and RelA on IAP expression. Expression of c-IAP1
was observed by Western blotting in A431-Neo cells stimulated with 10 ng/ml
EGF for 24 h and A431-RelA cells had a higher basal level of c-IAP1 that was
not further increased by EGF (Fig.
7). These results suggest that RelA up-regulates the expression of
c-IAP1 and EGF up-regulates it presumably through the activation of
NF-
B.

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FIG. 7. Effects of EGF on IAP expression in A431-Neo and A431-RelA cells.
Cells (1 x 106) were seeded in 35-mm Petri dishes and treated
with or without EGF (10 ng/ml) for 24 h. To detect IAP expression, the cell
lysates were resolved on 10% SDS-PAGE, electrotransferred onto a
nitrocellulose membrane, probed with c-IAP1 or -actin (control)
antibody, and detected by ECL as described under "Experimental
Procedures." The results were confirmed in another independent
experiment.
|
|
LY294002, an Inhibitor of PI-3K, Sensitizes A431-RelA Cells but Not
A431-Neo Cells to EGF-induced CytotoxicityWe also examined for the
involvement of the PI-3K pathway known to activate Akt that enables survival
of cells by the activation of NF-
B. In Western blot analysis using a
phosphorylation-specific antibody to Akt, there was no detectable
phosphorylation of Akt in A431-Neo cells in the presence or absence of EGF,
whereas even the unstimulated A431-RelA cells exhibited phosphorylation of Akt
that was further enhanced by EGF (Fig.
8A). An inhibitor of PI-3K, LY294002, inhibited
EGF-induced phosphorylation of Akt in A431-RelA cells whereas the expression
of total Akt protein remained unaffected by EGF treatment in A431-Neo and
A431-RelA cells (Fig.
8A). EGF decreased the growth of A431-Neo cells whereas
LY294002 alone did not affect their viability and addition of LY294002
together with EGF had no further effect on the growth of A431-Neo cells
(Fig. 8B). In
contrast, EGF did not inhibit the growth of A431-RelA cells but addition of
LY294002 together with EGF decreased their viability
(Fig. 8B). These
results suggest that RelA could activate Akt and EGF could activate it further
in A431-RelA cells and even though inhibition of PI-3K by LY294002 could not
enhance EGF-induced cell death of A431-Neo cells, it did sensitize A431-RelA
cells to EGF.

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FIG. 8. Effect of LY294002 on Akt phosphorylation and survival of A431-Neo and
A431-RelA cells. A, cells (1 x 106) grown in
35-mm Petri dishes were pretreated with LY294002 (20 µM) for 2 h
and then treated with EGF (10 ng/ml) for 1 h and the cells that received no
treatment served as control. Cell lysates were probed with phospho-Akt
(Thr308) antibody (1:1000) by Western blotting by ECL as described
above and the same blot was reprobed with an antibody (1:1000) to Akt.
B, cells were pretreated with LY294002 (20 µM) for 2 h
and then with EGF (10 ng/ml) and incubated for 24 h and the MTT assay was done
as described under "Experimental Procedures." The above
experiments were repeated with similar results.
|
|
Taken together, the results of the present study suggest that EGF induces
typical features of apoptosis in A431 cells such as the externalization of PS,
DNA fragmentation, and activation of caspases (3 and 8 but not 9). These
hallmarks of apoptosis induced by EGF were potentiated by NF-
B
inhibition by I
B-
and RelA relieved the effects of
I
B-
on EGF-mediated cytotoxicity. The chemical inhibitors of
NF-
B also enhanced the growth inhibitory effects of EGF and these
results together support an anti-apoptotic role for NF-
B. This is
further supported by the protective role of RelA observed in A431-RelA cells
against EGF-mediated apoptosis. A model for the regulation of EGF-induced
apoptosis incorporating the known mechanisms and contributions from the
present study is shown in Fig.
9.
 |
DISCUSSION
|
---|
Several studies have reported that high (nanomolar) concentrations of EGF
caused growth inhibition and apoptosis of A431 cells while low (picomolar)
concentrations of EGF promoted cell proliferation
(7,
38,
40,
43,
44). Many hypotheses have been
proposed to explain the mechanisms by which EGF inhibits cell proliferation,
arrests cell cycle, and induces apoptosis. Increase in EGFR tyrosine kinase
activity because of excessive ligand binding is said to result in growth
inhibition and programmed cell death and the level of EGFR tyrosine kinase
activity regulates the expression of p21
(17,
38). It has also been
suggested that EGF arrests cell cycle progression through Stat1 activation and
elevation of p21 that eventually inhibits cdk2 activity
(4345).
However, Stat1 activation by EGF was not detected in a number of cancer cell
lines with abnormal EGFR expression
(18,
45). The dual effect of EGF on
the proliferation of A431 cells is also attributed to differential activation
of p42 MAP kinase at low and high concentrations of EGF
(11,
46). Another hypothesis
proposes that EGF-induced apoptosis is the result of a decline in cell
adhesion (47), and detachment
of cells because of anoikis is said to be responsible for the paradoxical
anti-proliferative effects of EGF
(16,
48). Our results do not
support the view that EGF-induced apoptosis results from the loss of
cell-substratum attachment and many other workers also have not observed
EGF-induced anoikis of A431 or other EGFR-overexpressing cells
(8,
11,
49,
50). The results of the
present study suggest that EGF-induced apoptosis operates through the
extrinsic pathway mediated through caspase 8 and not the mitochondrial pathway
involving caspase 9. In contrast, EGF-induced apoptosis of MDA-MB-468 breast
cancer cells that also overexpress EGFR has been reported to involve the
mitochondrial pathway of caspase activation
(16). Similar to our results
with EGF, heregulin also enhanced the degradation of PARP but unlike EGF,
heregulin activated caspase 9 without any significant activation of caspase 3
(51). Cripto-1 mediates the
induction of caspase 3-like protease and down-regulates the expression of
Bcl-XL (15). UCVA-1 cells,
derived from human pancreas adenocarcinoma, have a high number of EGFRs but
their growth is not inhibited by EGF, highlighting the complexity of the
mechanisms involved in EGF-induced apoptosis
(52). Thus, the regulation of
EGF-induced apoptosis appears to be a complex process involving the interplay
of several regulatory molecules.
In addition to the mechanisms by which the growth factors exhibit both
stimulatory and inhibitory activity in a single cell, the final outcome is
presumably influenced by a host of regulatory molecules other than the growth
factors and their receptors
(53,
54). It is thus clearly
important to recognize that a potent mitogen like EGF also sends out apoptotic
signals and identify conditions in which these signals are regulated. In the
present study, we have hypothesized such a condition and demonstrated for the
first time that NF-
B inhibition makes A431 cells more susceptible to
EGF-induced apoptosis whereas RelA protects them against it. EGF stimulation
in A431 cells enhances the degradation of I
B-
, but not
I
B-
and proteasome inhibitors such as ALLN or MG132, block
EGF-mediated NF-
B activation, indicating that EGF-induced NF-
B
activation requires proteasome-dependent I
B degradation
(25). Similar to the findings
of the present study, EGF-induced DNA binding complex of NF-
B in A431
was found to be composed of p50/RelA heterodimers, but not c-Rel
(25). In agreement with our
results, a recent report has shown that both the protein kinase C inhibitor
Go6976 (also inhibits NF-
B) and expression of dominant-negative
NF-
B inhibitor kinase mutants caused apoptosis of EGF-stimulated
mammary tumor cells whereas EGF alone did not induce apoptosis in these cells
(55). In contrast to our
results, it has been reported that infection of adenoviral vectors expressing
I
B reversed the EGF-induced cell growth inhibition of A431 cells
(56). Use of high
concentrations of EGF (100 ng/ml), transient expression of I
B, and
drawing conclusions only based upon cell counting (proportion of live and dead
cells not shown) may account for this discrepancy. Furthermore, caspase
activities or other apoptotic parameters were not measured in that study.
Consistent with our results, blocking Ras (known to activate NF-
B) with
dominant negative Ras mutants in EGFR overexpressing cells also potentiated
EGF-induced apoptosis (8). EGF
did not affect the levels of several proteins that regulate apoptotic
pathways, including Bcl-2, Bcl-XL, Bax, and p53 in human ovarian cancer cells
(57). Many workers have shown
that Akt, a serine/threonine protein kinase and a downstream target of PI-3K,
suppresses apoptosis by activating NF-
B
(5860)
and accordingly blocking the EGF-induced phosphorylation of Akt sensitizes the
otherwise resistant A431-RelA cells to EGF-mediated growth inhibition.
However, the spontaneous phosphorylation of Akt in A431-RelA cells places Akt
as a downstream target of NF-
B and it is interesting to note that
blocking this basal phosphorylation with LY294002 does not affect their
viability. Consistent with our results, overexpression of RelA stimulated the
phosphorylation of Akt in NIH 3T3 cells and in addition increased the
expression of Akt mRNA and protein
(61). Akt does not seem to be
involved in EGF-induced NF-
B activation of MDA-MB-468 cells that also
overexpress EGFR (62). The
mechanism by which ectopic expression of RelA results in the phosphorylation
of Akt is not known and further work is in progress to understand the
contribution of Akt in EGF-induced apoptosis. Similar to the EGF-induced
expression of c-IAP1 in the present study, TNF also stimulated its expression
(63). Cbl-b inhibits
EGF-induced apoptosis by enhancing ubiquitination and degradation of activated
EGFR (64).
Our results support an anti-apoptotic role for NF-
B and suggest the
possibility that EGF-induced apoptosis in vivo may be regulated by
NF-
B. However, whether EGF-induced apoptosis occurs in vivo in
tumor cells is not known. If EGF-induced apoptosis does not occur in
vivo, it is still logical to think that anti-apoptotic factors such as
NF-
B guard the tumor cells against responding to such apoptotic
stimuli. Consistent with this speculation, NF-
B is constitutively
activated in a number of tumors
(22,
26). In addition, tumor cells
are also known to express many anti-apoptotic proteins such as Bcl-2 and their
role in EGF-induced apoptosis remains to be determined. Our identification of
a role for NF-
B in EGF-induced apoptosis opens up new lines of
investigation pertaining to EGF receptor-mediated signaling. Further studies
on the role of NF-
B and its regulators in EGF-induced apoptosis are
likely to be relevant for a better understanding of the biology of human
tumors as well as EGF-induced normal growth.
 |
FOOTNOTES
|
---|
* This work was supported in part by grants from the Department of Science
and Technology, Government of India and the Science, Technology and
Environment committee, Government of Kerala, India (to D. K.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Supported by a Senior Research Fellowship from the Council of Scientific
and Industrial Research, India. 
To whom correspondence should be addressed. Tel.: 91-471-2347975; Fax:
91-471-2348096; E-mail:
dkarunagaran{at}hotmail.com.
1 The abbreviations used are: EGF, epidermal growth factor; ECL, enhanced
chemiluminescence; EGFR, epidermal growth factor receptor; EMSA,
electrophoretic mobility shift assay; HA, hemagglutinin; I
B, inhibitor
B; IKK, I
B kinase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-
B,
nuclear factor
B; PARP, poly-(ADP-ribose) polymerase; PBS,
phosphate-buffered saline; PI, propidium iodide; PS, phosphatidylserine; TNF,
tumor necrosis factor; IAP, inhibitor of apoptosis protein; PI-3K,
phosphatidylinositol 3-kinase; AFC, 7-amino-4-trifluromethyl coumarin; DTT,
dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Yosef Yarden for providing EGFR antibody, Sudhir Krishna for
HA-tagged pcDNA3-I
B-
construct, Paul Chiao for relA in
pMT2T vector, and Bava Smitha and Goodwin Jinesh for technical help.
 |
REFERENCES
|
---|
- Grant, S., Qiao, L., and Dent, P. (2002)
Front. Biosci. 7,
376389
- Yarden, Y. (2001) Eur. J.
Cancer 37, Suppl. 4,
S3S8
- Schlessinger, J. (2000) Cell
103,
211225[Medline]
[Order article via Infotrieve]
- Gullick, W. J. (1991) Br. Med.
Bull. 47,
8798[Abstract]
- Harris, A. L. (1994) Breast Cancer Res.
Treat. 29,
12[Medline]
[Order article via Infotrieve]
- Bartlett, J. M., Langdon, S. P., Simpson, B. J., Stewart, M.,
Katsaros, D., Sismondi, P., Love, S., Scott, W. N., Williams, A. R., Lessells,
A. M., Macleod, K. G., Smyth, J. F., and Miller, W. R. (1996)
Br. J. Cancer 73,
301306[Medline]
[Order article via Infotrieve]
- Barnes, D. W. (1982) J. Cell
Biol. 93,
14[Abstract]
- Hognason, T., Chatterjee, S., Vartanian, T., Ratan, R. R.,
Ernewein, K. M., and Habib, A. A. (2001) FEBS
Lett. 491,
915[CrossRef][Medline]
[Order article via Infotrieve]
- Daly, J. M., Olayioye, M. A., Wong, A. M., Neve, R., Lane, H. A.,
Maurer, F. G., and Hynes, N. E. (1999)
Oncogene 18,
34403451[CrossRef][Medline]
[Order article via Infotrieve]
- Xi, Q. S., Pan, W., Zhang, Q., Qian, X. G., Li, Z. P., and Gan, R.
B. (2000) Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao
(Shanghai) 32,
601604[Medline]
[Order article via Infotrieve]
- Silvy, M., Martin, P. M., Chajry, N., and Berthois, Y.
(1998) Endocrinology
139,
23822391[Abstract/Free Full Text]
- Iwamoto, R., Handa, K., and Mekada, E. (1999)
J. Biol. Chem. 274,
2590625912[Abstract/Free Full Text]
- Conner, E. A., Teramoto, T., Wirth, P. J., Kiss, A., Garfield, S.,
and Thorgeirsson, S. S. (1999)
Carcinogenesis 20,
583590[Abstract/Free Full Text]
- Kim, H. R., Upadhyay, S., Li, G., Palmer, K. C., and Deuel, T. F.
(1995) Proc. Natl. Acad. Sci. U. S. A.
92,
95009504[Abstract]
- De Santis, M. L., Martinez-Lacaci, I., Bianco, C., Seno, M.,
Wallace-Jones, B., Kim, N., Ebert, A., Wechselberger, C., and Salomon, D. S.
(2000) Cell Death Differ.
7,
189196[CrossRef][Medline]
[Order article via Infotrieve]
- Kottke, T. J., Blajeski, A. L., Martins, L. M., Mesner, P. W., Jr.,
Davidson, N. E., Earnshaw, W. C., Armstrong, D. K., and Kaufmann, S. H.
(1999) J. Biol. Chem.
274,
1592715936[Abstract/Free Full Text]
- Reddy, K. B., Keshamouni, V. G., and Chen, Y. Q.
(1999) Int. J. Oncol.
15,
301306[Medline]
[Order article via Infotrieve]
- Chin, Y. E., Kitagawa, M., Kuida, K., Flavell, R. A., and Fu, X. Y.
(1997) Mol. Cell. Biol.
17,
53285337[Abstract]
- Strasser, A., O'Connor, L., and Dixit, V. M. (2000)
Annu. Rev. Biochem. 69,
217245[CrossRef][Medline]
[Order article via Infotrieve]
- Igney, F. H., and Krammer, P. H. (2002)
Nat. Rev. Cancer 2,
277288[CrossRef][Medline]
[Order article via Infotrieve]
- Sun, X. M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R.,
and Cohen, G. M. (1999) J. Biol. Chem.
274,
50535060[Abstract/Free Full Text]
- Karin, M., Cao, Y., Greten, F. R., and Li, Z. W.
(2002) Nat. Rev. Cancer
2,
301310[CrossRef][Medline]
[Order article via Infotrieve]
- Obata, H., Biro, S., Arima, N., Kaieda, H., Kihara, T., Eto, H.,
Miyata, M., and Tanaka, H. (1996) Biochem. Biophys.
Res. Commun. 224,
2732[CrossRef][Medline]
[Order article via Infotrieve]
- Biswas, D. K., Cruz, A. P., Gansberger, E., and Pardee, A. B.
(2000) Proc. Natl. Acad. Sci. U. S. A.
97,
85428547[Abstract/Free Full Text]
- Sun, L., and Carpenter, G. (1998)
Oncogene 16,
20952102[CrossRef][Medline]
[Order article via Infotrieve]
- Rayet, B., and Gelinas, C. (1999)
Oncogene 18,
69386947[CrossRef][Medline]
[Order article via Infotrieve]
- Beg, A. A., and Baltimore, D. (1996)
Science 274,
782784[Abstract/Free Full Text]
- Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and
Verma, I. M. (1996) Science
274,
787789[Abstract/Free Full Text]
- Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr.
(1996) Science
274,
784787[Abstract/Free Full Text]
- Cusack, J. C., Jr., Liu, R., Houston, M., Abendroth, K., Elliott,
P. J., Adams, J., and Baldwin, A. S., Jr. (2001)
Cancer Res. 61,
35353540[Abstract/Free Full Text]
- Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J., Malim, M. H.,
and Ballard, D. W. (1997) Proc. Natl. Acad. Sci. U. S.
A. 94,
1005710062[Abstract/Free Full Text]
- You, M., Ku, P. T., Hrdlickova, R., and Bose, H. R., Jr.
(1997) Mol. Cell. Biol.
17,
73287341[Abstract]
- Anto, R. J., Maliekal, T. T., and Karunagaran, D.
(2000) J. Biol. Chem.
275,
1560115604[Abstract/Free Full Text]
- Bours, V., Burd, P. R., Brown, K., Villalobos, J., Park, S.,
Ryseck, R. P., Bravo, R., Kelly, K., and Siebenlist, U. (1992)
Mol. Cell. Biol. 12,
685695[Abstract]
- Taylor, N. A., and Docherty, K. (1996) in
Gene Transcription: RNA Analysis (Docherty, K., ed)
pp. 9697, John Wiley & Sons Ltd.,
London
- Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L.
(1988) Exp. Cell Res.
175,
184191[Medline]
[Order article via Infotrieve]
- Ito, A., Uehara, T., Tokumitsu, A., Okuma, Y., and Nomura, Y.
(1999) Biochim. Biophys. Acta
1452,
263274[Medline]
[Order article via Infotrieve]
- Gulli, L. F., Palmer, K. C., Chen, Y. Q., and Reddy, K. B.
(1996) Cell Growth Differ.
7,
173178[Abstract]
- Filmus, J., Pollak, M. N., Cailleau, R., and Buick, R. N.
(1985) Biochem. Biophys. Res. Commun.
128,
898905[Medline]
[Order article via Infotrieve]
- Gill, G. N., and Lazar, C. S. (1981)
Nature 293,
305307[Medline]
[Order article via Infotrieve]
- Homburg, C. H., de Haas, M., von dem Borne, A. E., Verhoeven, A.
J., Reutelingsperger, C. P., and Roos, D. (1995)
Blood 85,
532540[Abstract/Free Full Text]
- Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger,
C. (1995) J. Immunol. Methods
184,
3951[CrossRef][Medline]
[Order article via Infotrieve]
- Fan, Z., Lu, Y., Wu, X., DeBlasio, A., Koff, A., and Mendelsohn, J.
(1995) J. Cell Biol.
131,
235242[Abstract]
- Jakus, J., and Yeudall, W. A. (1996)
Oncogene 12,
23692376[Medline]
[Order article via Infotrieve]
- Chin, Y. E., Kitagawa, M., Su, W. C., You, Z. H., Iwamoto, Y., and
Fu, X. Y. (1996) Science
272,
719722[Abstract]
- Chajry, N., Martin, P. M., Pages, G., Cochet, C., Afdel, K., and
Berthois, Y. (1994) Biochem. Biophys. Res.
Commun. 203,
984990[CrossRef][Medline]
[Order article via Infotrieve]
- Cao, L., Yao, Y., Lee, V., Kiani, C., Spaner, D., Lin, Z., Zhang,
Y., Adams, M. E., and Yang, B. B. (2000) J. Cell.
Biochem. 77,
569583[CrossRef][Medline]
[Order article via Infotrieve]
- Genersch, E., Schneider, D. W., Sauer, G., Khazaie, K., Schuppan,
D., and Lichtner, R. B. (1998) Int. J.
Cancer 75,
205209[CrossRef][Medline]
[Order article via Infotrieve]
- Lee, K., Tanaka, M., Hatanaka, M., and Kuze, F. (1987)
Exp. Cell Res. 173,
156162[Medline]
[Order article via Infotrieve]
- Dong, X. F., Berthois, Y., and Martin, P. M. (1991)
Anticancer Res. 11,
737743[Medline]
[Order article via Infotrieve]
- Le, X. F., Marcelli, M., McWatters, A., Nan, B., Mills, G. B.,
O'Brian, C. A., and Bast, R. C., Jr. (2001)
Oncogene 20,
82588269[CrossRef][Medline]
[Order article via Infotrieve]
- Hirai, M., Kobayashi, M., and Shimizu, N. (1990)
Cell. Signalling 2,
245252[CrossRef][Medline]
[Order article via Infotrieve]
- Sporn, M. B., and Roberts, A. B. (1988)
Nature 332,
217219[CrossRef][Medline]
[Order article via Infotrieve]
- Lehto, V. P. (2001) FEBS Lett.
491,
13[CrossRef][Medline]
[Order article via Infotrieve]
- Biswas, D. K., Martin, K. J., McAlister, C., Cruz, A. P., Graner,
E., Dai, S. C., and Pardee, A. B. (2003) Cancer
Res. 63,
290295[Abstract/Free Full Text]
- Ohtsubo, M., Takayanagi, A., Gamou, S., and Shimizu, N.
(2000) J. Cell. Physiol.
184,
131137[CrossRef][Medline]
[Order article via Infotrieve]
- Cenni, B., Aebi, S., Nehme, A., and Christen, R. D.
(2001) Cancer Chemother. Pharmacol.
47,
397403[CrossRef][Medline]
[Order article via Infotrieve]
- Romashkova, J. A., and Makarov, S. S. (1999)
Nature 401,
8690[CrossRef][Medline]
[Order article via Infotrieve]
- Pianetti, S., Arsura, M., Romieu-Mourez, R., Coffey, R. J., and
Sonenshein, G. E. (2001) Oncogene
20,
12871299[CrossRef][Medline]
[Order article via Infotrieve]
- Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer,
L. M., and Donner, D. B. (1999) Nature
401,
8285[CrossRef][Medline]
[Order article via Infotrieve]
- Meng, F., Liu, L., Chin, P. C., and D'Mello, S. R.
(2002) J. Biol. Chem.
277,
2967429680[Abstract/Free Full Text]
- Habib, A. A., Chatterjee, S., Park, S. K., Ratan, R. R., Lefebvre,
S., and Vartanian, T. (2001) J. Biol.
Chem. 276,
88658874[Abstract/Free Full Text]
- Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and
Baldwin, A. S., Jr. (1998) Science
281,
16801683[Abstract/Free Full Text]
- Ettenberg, S. A., Rubinstein, Y. R., Banerjee, P., Nau, M. M.,
Keane, M. M., and Lipkowitz, S. (1999) Mol. Cell.
Biol. Res. Commun. 2,
111118[CrossRef][Medline]
[Order article via Infotrieve]