From the Equipe Facteurs de Croissance, UPRES
EA-1033 Biologie du Développement, Université des Sciences
et Technologies de Lille, 59655 Villeneuve d' ASCQ France, the
¶ Immunopathologie Cellulaire des Maladies Infectieuses, CNRS, UMR
8527, Institut de Biologie de Lille, 59000 France, the ** Department of
Anatomical Sciences, University of Queensland, St. Lucia,
Queensland 4072, Australia, and the
Laboratoire
d'Oncologie Moléculaire Humaine, Centre Oscar Lambret, 59020 Lille, France
Received for publication, November 20, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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We show here that the neurotrophin nerve growth
factor (NGF), which has been shown to be a mitogen for breast cancer
cells, also stimulates cell survival through a distinct
signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and
MDA-MB-231) were found to express both types of NGF receptors:
p140trkA and p75NTR. The two other tyrosine
kinase receptors for neurotrophins, TrkB and TrkC, were not expressed.
The mitogenic effect of NGF on breast cancer cells required the
tyrosine kinase activity of p140trkA as well as the
mitogen-activated protein kinase (MAPK) cascade, but was
independent of p75NTR. In contrast, the anti-apoptotic
effect of NGF (studied using the ceramide analogue C2) required
p75NTR as well as the activation of the transcription
factor NF-kB, but neither p140trkA nor MAPK was necessary.
Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival,
although not proliferation, emphasizing the importance of
p75NTR in NGF-mediated survival. Both the pharmacological
NF- Nerve growth factor
(NGF)1 is the archetypal
member of the neurotrophin superfamily, which also includes
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),
NT-4/5, and NT-6 (1). NGF interacts with two classes of membrane
receptor: the TrkA proto-oncogene product p140trkA, which
possesses intrinsic tyrosine kinase activity, and a secondary receptor,
p75NTR, that belongs to the tumor necrosis factor (TNF)
receptor family (2). The stimulation of cell survival and cell
differentiation by NGF and other neurotrophins have been described
primarily in neuronal cell systems (3). Although the neurotrophic
effect through p140trkA is known to involve the MAPK cascade,
the role of p75NTR is still controversial; there is
evidence that it can both positively and negatively regulate neuronal
cell death and differentiation, depending on the cell type examined
(4). In some cases, p75NTR is an inducer of apoptosis, even
without NGF stimulation (5), whereas in other cases the activation of
p75NTR by NGF results in a protection from cell death (6).
In addition to its neurotrophic function, other activities of NGF have
been described. For example, NGF can modulate gene expression in
monocytes (7), it is chemotactic for melanocytes (8), and its
inhibition on p75NTR can block the migration of Schwann
cells (9). NGF also stimulates the proliferation of chromaffin cells
(10), lymphocytes (11), and keratinocytes (12). We have previously
shown that NGF is mitogenic for cancerous but not normal human breast
cells (13), and these data, as well as others showing a role for NGF in
the stimulation of prostatic cancer cells (14-17), implicate NGF in non-neuronal carcinogenesis.
Both cellular proliferation as well as tumor cell survival
are crucial for malignant progression. The effect of NGF on the survival of cancer cells through the p75NTR receptor has
been shown for neuroblastoma (18) and schwannoma (6). In prostate
cancer, p75NTR has been shown to be a mediator of NGF's
effects during critical phases of developmental cell death and
carcinogenic progression (19). To date only the mitogenic effect of NGF
for breast cancer cells has been described (13), with its roles in the
control of breast cancer cell survival unknown.
In this study, we have shown that, in addition to its mitogenic effect,
NGF is also an anti-apoptotic factor for breast cancer cells. These
cells express mRNA for both p140trkA and p75NTR
receptors. Our results indicate that the mitogenic effect of NGF
requires p140trkA and the MAPK cascade, but not the
p75NTR receptor, whereas the promotion of cell survival
strictly requires p75NTR as well as NF- Materials--
Cell culture reagents were purchased from
BioWhittaker (France) except insulin, which was obtained from Organon
(France). Recombinant human nerve growth factor, brain
derived growth factor (BDNF), and neurotrophins 3 (NT-3) and 4 (NT-4)
were from R & D Systems (UK). K-252a (inhibitor of trk-tyrosine kinase
activity) and PD98059 (inhibitor of MAPK cascade) were from Calbiochem
(France). The mouse monoclonal anti-NGF receptor (p75NTR)
antibody was from Euromedex (France) and was previously described for
its ability to block the interaction between p75NTR and NGF
(20). The anti-lamin B (C-20), goat polyclonal IgG, and the polyclonal
anti-p140trkA (trk763) were from Santa Cruz Biotechnology. C2
ceramide analogue (N-acetyl-D-sphingosine),
Hoechst 33258, and electrophoresis reagents were from Sigma Chemical
Co. (France). The SN50 NF- Cell Culture--
Breast cancer cell lines (MCF-7, T47-D, BT-20,
and MDA-MB-231) were obtained from the American Type Culture Collection
and routinely grown as monolayer cultures. Cells were maintained in minimal essential medium (Earle's salts) supplemented with 20 mM Hepes, 2 g/liter sodium bicarbonate, 2 mM
L-glutamine, 10% fetal calf serum (FCS), 100 units/ml
penicillin-streptomycin, 50 µg/ml gentamicin, 1% of non-essential
amino acids, and 5 µg/ml insulin.
Detection of Neurotrophin Receptors mRNA Expression--
The reverse transcription reaction mixture contained 2 g of
purified total RNA (extracted from breast cancer cell lines, NT-2 cells, or SY5Y cells), 1× reverse transcription reaction buffer, 10 mM DTT, 400 mM dNTP each, 2.5 M oligo(dT) 18 primer, 40 units of RNasin, and 200 units of
Moloney murine leukemia virus reverse transcriptase were added to 25 µl of total reaction volume. All the reaction mixtures were incubated
at 37 °C for 1 h and then inactivated at 95 °C for 5 min.
Polymerase chain reaction was performed on cDNAs after RT or
corresponding total RNA samples without the RT step for negative
controls. The primers used for trkA and p75 RT-PCR detection
in breast cancer cell lines were as follows: trkA sense
primer, 5' (291)-CATCGTGAAGAGTGTCTCCG-3' (311) and antisense primer, 5'
(392)-GAGAGAGACTCCAGAGCGTTGAA-3' (370) or p75 sense primer, 5'
(442)-CCTACGGCTACTACCAGGATGAG-3' (462) and antisense primer, 5'
(588)-TGGCCTCGTCGGAATACG-3' (571). The primers used for RT-PCR
comparative detection of trks in MCF-7 cells were as follows:
trkA sense primer, 5' (118)-AGGCGGTCTGGTGACTTCGTTG-3' (139)
and antisense primer, 5' (1162)-GGCAGCCAGCAGGGTGTAGTTC-3' (1141) or
trkB sense primer, 5' (134)-CGAGGTTGGAACCTAACAGCATTG-3' (157) and
antisense primer, 5' (1182)-GTCAGTTGGCGTGGTCCAGTCTTC-3' (1159)
or trkC sense primer, 5' (219)-CACGGACATCTCAAGGAAGAGCA-3' (241) and
antisense primer, 5' (1078)-CTGAGAACTTCACCCTCCTGGTAG-3' (1056).
Each pair of primers was used in RT-PCR reaction to amplify trks or
p75. To PCR tubes were added 5 µl of PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 10 µl of 15 mM
MgCl2, 1 µl of 10 mM dNTP mix, 1 µl of
cDNA or total mRNA (for negative control), 1 µl of 50 mM respective primers, 1 µl of 2.5 units/µl
Taq DNA polymerase, and water to a total volume of 50 µl.
The PCR conditions were as follows: after 95 °C for 3 min for
denaturing cDNA, 30 cycles were run at 94 °C for 1 min, 57 °C
for 2 min, and 72 °C for 3 min. The PCR tubes were incubated for a
further 10 min at 72 °C for the extension of cDNA fragments
after the final cycle, and the PCR products were electrophoresed in an
agarose gel.
Cell Growth Assay--
Experiments were performed as previously
described (13). 35-mm diameter dishes were inoculated with 2 × 104 cells/dish in 2 ml of medium containing 10% FCS. After
24 h, cells were washed twice with serum-free medium. Next day,
the medium was replaced with 2 ml of serum free medium containing 100 ng/ml NGF or various concentrations of other neurotrophins (BDNF, NT-3,
NT-4/5). To study the effect of pharmacological inhibitors or blocking
antibodies, various concentrations were added simultaneously with NGF
(100 ng/ml). After 2 days of NGF exposure, cells were harvested by
trypsinization and counted using an hemocytometer.
Determination of the Percentage of Apoptotic Cell
Nuclei--
Apoptosis of breast cancer cells was induced by the
ceramide analogue C2, which has been described as a pro-apoptotic agent for human breast cancer cells (21, 22). Apoptosis was obtained by
treatment with 2 µM C2 for 24 h. To evaluate the
anti-apoptotic activity of NGF, various concentrations of this factor
were tested; we found that the maximal effect was obtained for 100 ng/ml. Consequently, this concentration was used in all experiments
with pharmacological inhibitors or blocking antibody. For determination
of apoptotic cell percentage, cells were fixed with cold methanol
( Statistical Analysis and Software--
The statistical analysis
of the data gathered from cell and apoptotic nuclei counting was
performed using SPSS version 9.0.1 (SPSS inc., Chicago, IL). Analyses
of variance were followed by the Tukey's test to determine the significance.
NGF Receptors and PARP Immunoblotting--
Subconfluent cell
cultures were harvested by scraping in serum-free medium. After
centrifugation (1000 × g, 5 min), the pellet was
treated with lysis buffer (0.3% SDS, 200 mM
dithiothreitol) and boiled 5 min. In the case of PARP, the pellet was
lysed with urea-rich buffer (62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS), sonicated and incubated at
65 °C for 15 min. The lysates were subjected to SDS-PAGE,
transferred onto a nitrocellulose membrane (Immobilon-P, Millipore) by
electroblotting (100 V, 75 min), and probed with anti-trkA,
anti-p75NTR or anti-PARP antibodies at 4 °C overnight.
The membranes were then incubated at room temperature for 3 h with
biotin-conjugated anti-rabbit (TrkA) or anti-mouse (p75NTR
and PARP) immunoglobulin G. After 1 h of incubation with
extravidin, the reaction was revealed using the chemiluminescence kit
ECL (Amersham Pharmacia Biotech) with Kodak X-Omat AR film.
Detection of p140trkA and MAPK Activation--
Proteins
were extracted in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 0.1% SDS, 1% Nonidet P-40, 100 µM sodium orthovanadate) prior to immunoprecipitation.
Preclearing was done with protein A-agarose (10 µl/250 µl, 60 min,
4 °C). After centrifugation (10,000 × g, 2 min),
the supernatant was incubated with monoclonal anti-MAPK (anti-ERK2)
antibody (10 µl/250 µl, 60 min, 4 °C). Protein A-agarose (10 µl) was added for 60 min (4 °C) and then pelleted by
centrifugation (10,000 × g, 2 min). The pellet was
then rinsed three times with lysis buffer and boiled for 5 min in
Laemmli buffer. After SDS-PAGE and electroblotting, nitrocellulose
membranes were blocked with 3% bovine serum albumin. Membranes were
then incubated with PY20 anti-phosphotyrosine antibody overnight at
4 °C, rinsed, and incubated with a horseradish peroxidase-conjugated
anti-mouse IgG for 3 h at room temperature. Membranes were rinsed
overnight at 4 °C before visualization with ECL.
Cell Fractionation and NF- Transfection of I NGF Mitogenic and Anti-apoptotic Activity for Breast Cancer
Cells--
The effects of 100 ng/ml NGF on cell proliferation and
C2-induced apoptosis were evaluated by cell counting and Hoechst
staining, respectively. The results show that NGF induces an increase
in cell number for all breast cancer cell lines tested (Fig.
1A). We have previously
demonstrated that NGF has a direct mitogenic effect on breast cancer
cells by recruiting cells in G0 phase and by
shortening the G1 length (Descamps et al.,
1998). In addition, NGF rescued breast cancer cells undergoing
C2-induced apoptosis; the maximum survival was observed at 200 ng/ml
(Fig. 1B). The morphology of cells undergoing this
NGF-induced anti-apoptotic rescue was quite distinct (Fig.
2A). The induction of
apoptosis by C2 was found to involve cleavage of poly(A)DP-ribose
polymerase (PARP); this cleavage was reversed by NGF (Fig.
2B).
TrkA and p75NTR Expression--
RT-PCR was used to
show the expression of mRNA for both high and low affinity NGF
receptors in MCF-7, T47-D, BT-20, and MDA-MB-231 cells (Fig.
3A); the 102-bp band for the
TrkA transcript and a 147-bp band for the p75NTR transcript
were readily detectable on 1% agarose gels. Moreover, Western blotting
demonstrated that both p140trkA and p75NTR were
present in all the breast cancer cell lines (Fig. 3B).
Real-time quantitative RT-PCR indicated that there was no significant
change in the levels of TrkA and p75NTR mRNAs in the
presence of FCS, NGF, or C2 (data not shown) and that the levels of
mRNA for TrkA and p75NTR in breast cancer cells was
between 5 and 10 times lower than the level observed in SY5Y
neuroblastoma cells (data not shown). This indicates that NGF receptor
expression in breast cancer cells is relatively limited. It should be
noticed that, although mRNA levels of NGF receptors differ between
breast cancer cells and SY5Y, the protein levels apparently do not.
However, it has been shown before that the level of a given cellular
protein cannot be simply deduced from mRNA transcript level (24).
One could hypothesize that the stability of mRNA and/or protein for
NGF receptors, differs between breast cancer cells and neuroblastoma cells, leading to the observed disproportionality between mRNA and
protein levels.
Involvement of p140trkA and p75NTR in Mitogenic
and Survival Activities of NGF--
We used a combination of specific
antibodies and pharmacological inhibitors to study the putative
functions of p140trkA and p75NTR in the stimulation
of proliferation and cell survival induced by NGF. The Trk tyrosine
kinase inhibitor K-252a, and the MEK inhibitor PD98059, both strongly
inhibited the growth-stimulatory effect of NGF on MCF-7 cells, but had
no effect on its anti-apoptotic effects (Fig.
4). Conversely, neither the
anti-p75NTR blocking antibody nor the NF- NF- This study shows that, in addition to its mitogenic activity, NGF
is anti-apoptotic for breast cancer cells, and that these two
biological effects are differentially mediated by the p140trkA
and p75NTR receptors, respectively. The growth of breast
cancer results from a balance between cell proliferation and apoptosis,
both of which can be modulated by various regulatory peptides. For example, epidermal growth factor, fibroblast growth factors, and insulin-like growth factor-1 can all stimulate the proliferation and
survival of breast cancer cells (26). On the other hand, agents such as
transforming growth factor- The function of NGF as a survival factor has been extensively described
for neurons in both in vitro and in vivo (32).
However, the intracellular signaling involved in the anti-apoptotic
activity of NGF in neurons remains controversial. The
p140trkA/MAPK cascade is generally described as protective for
neuronal cell death, although there has been a recent report of a novel apoptotic pathway mediated by p140trkA/MAPK in medulloblastoma
cells (33). Unlike the p140trkA receptors, the definition of
the precise physiological role of p75NTR has proven
difficult (4). The p75NTR receptor belongs to the
TNF-receptor family, including among others, types I and II of the TNF
receptor, the Fas antigen, and CD40 (34). The common cellular responses
to activation of this family of receptors are the activation of gene
transcription via nuclear factor-kB (NF- In conclusion, our results demonstrate that NGF is an anti-apoptotic
factor for human breast cancer cells and that the signaling pathway
leading to this survival activity is distinct from the signaling
pathway, which leads to mitogenic stimulation. Although p140trkA and the MAPKs mediate the mitogenic activity of NGF,
its anti-apoptotic activity required p75NTR and NF-B inhibitor SN50, and cell transfection with IkBm, resulted in a
diminution of NGF anti-apoptotic effect. These data show that two
distinct signaling pathways are required for NGF activity and confirm
the roles played by p75NTR and NF-
B in the activation of
the survival pathway in breast cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, but not
p140trkA and MAPK. Thus the mitogenic and anti-apoptotic
effects of NGF on breast cancer cells are mediated through two
different signaling pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B inhibitor peptide, the rabbit polyclonal
anti-NF-
B p65 antibody, was obtained from TEBU (France). Anti-PARP
antibody was from Oncogene Research Products (UK). Primers and probes
for TrkA and p75NTR, probe for TATA box binding protein
(TBP) were from Eurogentec (Belgium). RT-PCR reagents were from Applied
Biosystems (France). Lipofectin reagent and Opti-MEM were provided by
Life Technologies, Inc. (France). The green fluorescence protein
plasmid (EGFPC1) was purchased from CLONTECH, and
the dominant-negative I
B
mutant (I
Bm) expression vectors (in
PCDNA3) containing a Ser to Ala substitution at residues 32 and 36 were obtained from Dr. Jean Feuillard (UPRES EA 1625, Bobigny,
France). p65 (rel-A) and c-rel cDNA were cloned at EcoRI
site in PSVK3 expression plasmid. All vectors were obtained from Dr.
Pascale Crépieux (McGill University, Montreal). The SY5Y subclone
of SK-N-SH neuroblastoma cell line was a kind gift of Dr. Luc
Buée (INSERM, U422, Lille, France). NT-2 (Ntera/D1) human neural
precursor cells (Stratagene) are derived from a clone of the NT-2 teratocarcinoma.
20 °C) for 10 min and washed twice with phosphate-buffered saline
(PBS) before staining with 1 µg/ml Hoechst 33258 for 10 min at room temperature in the dark. Cells were then washed with PBS and mounted with coverslips using Glycergel (Dako). The apoptotic cells
exhibiting condensed and fragmented nuclei were counted under an
Olympus-BH2 fluorescence microscope in randomly selected fields. A
minimum of 500-1000 cells was examined for each condition, and results were expressed as a ratio of the total number of cells counted.
B Detection--
Cell nuclear
extracts were prepared as described by Herrmann et al. (23).
Cells were trypsinized and then pelleted in minimal essential medium
containing 10% FCS. After washing with ice-cold PBS, cells were
repelleted and resuspended in 400 µl of ice-cold hypotonic buffer (10 mM Hepes, pH 7.8, 10 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA, 10 µg/ml aprotinin, 0.5 µg/ml leupeptin, 3 mM phenylmethylsulfonyl fluoride, and
3 mM DTT). After 10 min on ice, 25 µl of 10% Nonidet P-40 was added and crude nuclei were collected by centrifugation for 5 min. The nuclear pellet was resuspended in high salt buffer (50 mM Hepes, pH 7.4, 50 mM KCl, 300 mM
NaCl, 0.1 mM EDTA, 10% (v/v) glycerol, 3 mM
DTT, and 3 mM phenylmethylsulfonyl fluoride). After 30 min
on ice with frequent agitation, the insoluble nuclear material was
pelleted in a microcentrifuge for 10 min. Crude nuclear protein
was collected from the supernatant and snap-frozen in a dry ice/ethanol
bath. After thawing and boiling for 5 min in Laemmli buffer, the
nuclear extracts were subjected to SDS-PAGE and probed with an
anti-NF-
B p65 antibody. A control was established with anti-lamin B antibody.
, c-rel, and rel-A--
Cotransfection
experiments were carried out using Lipofectin reagent, as described by
the manufacturer. Briefly, MCF-7 cells were incubated for 5 h in 1 ml of Opti-MEM transfection medium containing 8 µl of Lipofectin
reagent, 0.8 µg of green fluorescence protein (GFP)-carrying vector
and 0.2 µg of empty vector PCDNA3 or 0.2 µg of I
Bm. In the
case of c-rel or rel-A, cells were cotransfected with 0.8 µg of
GFP-carrying GFP and 0.6 µg of PSVK3 (empty plasmid), c-rel, or rel-A. Cells were then grown for 24 h with 10% FCS
minimal essential medium and rinsed for 2 h in serum-free medium
before incubation in serum-free medium in the presence or absence of 100 ng/ml NGF and/or 2 µM C2 for another 24 h. Cells
were then fixed with paraformaldehyde 4% (4 °C) for 30 min, and the
percentage of apoptotic cell nuclei in GFP-stained cells was determined
as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of NGF on the growth and survival of
breast cancer cells. A, breast cancer cells were
serum-deprived in minimum essential medium, and after 24 h the NGF
(100 ng/ml) was added. After 48 h, cells were harvested and
counted. B, cells were serum-deprived in minimum essential
medium and treated with 2 µM C2 with or without 100 ng/ml
NGF. After 24 h, cells were fixed and the proportion of apoptotic
nuclei were determined after Hoechst staining under an Olympus-BH2
fluorescence microscope. For measurement of both cell number and
apoptosis, results are expressed as the means ± S.D. of five
separate experiments. Significance was determined using the Tukey's
test (*, p < 0.01).
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Fig. 2.
Anti-apoptotic effect of NGF.
A, Hoechst staining of apoptotic cell nuclei in control, C2
and C2+NGF-treated MCF-7 cells. Cells were serum-deprived in minimum
essential medium and treated with C2. NGF was added at 100 ng/ml. After
24 h, cells were fixed and apoptotic nuclei were observed after
Hoechst staining. B, immunoblot detection of PARP cleavage.
C2-induced PARP cleavage was reversed by p75NTR activation
mediated by NGF. MCF-7 cells were serum-deprived in minimum essential
medium for 24 h and were then treated with 100 ng/ml NGF in the
presence or absence of 2 µM C2, 10 nM K-252a,
10 µM PD98059, or 10 µg/ml
anti-p75NTR-blocking antibody (Euromedex) for another 24-h
period. Proteins were detected after SDS-PAGE of cell preparations from
MCF-7 breast cancer cells, electroblotting onto nitrocellulose, and
immunodetection with anti-PARP antibodies.
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Fig. 3.
TrkA and p75NTR expression in
breast cancer cells. A, agarose gel electrophoresis of
RT-PCR products evidenced a 102-bp band and a 147-bp band, which are
characteristic of TrkA and p75NTR, respectively. Both NGF
receptors were found in all cell types tested. B,
p140trkA and p75NTR were immunodetected after
SDS-PAGE of breast cancer cell lines. The neuroblastoma cells SY5Y were
used as positive control for the expression of NGF receptors.
B inhibitor
SN50 affected NGF-stimulated proliferation, although both strongly
reduced the anti-apoptotic effects (Fig. 4). The tyrosine kinase
activity of p140trkA was inhibited by K-252a but not by the
anti-p75NTR or PD98059 (Fig.
5). On the other hand, the activity of
the MAPKs was inhibited by K-252a and PD98059 but not by the
anti-p75NTR (Fig. 5). It should be noted that the SN50
peptidic inhibitor of NF-
B, similarly to the
anti-p75NTR, inhibited the anti-apoptotic effect of NGF
but neither its proliferative effect nor its activation of
p140trkA and MAPKs. The effect of other neurotrophins on MCF-7
cell growth and survival was also evaluated (Fig.
6A). In contrast to NGF, no
proliferative effect was provided by BDNF, NT-3, or NT-4/5. However,
all neurotrophins tested exhibited a rescue effect on C2-treated cells
that was not altered in the presence of the trk inhibitor K-252a (Fig.
6B). These data suggest that trk receptors are not involved
in NGF survival activity. Moreover, the participation of trkB and trkC
in these events can be ruled out, because they are not expressed in
these breast cancer cells (Fig. 6C).
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Fig. 4.
Pharmacological modulation of the
proliferative and anti-apoptotic effect of NGF. MCF-7 cells were
starved in minimum essential medium, and after 24 h, 100 ng/ml NGF
was added with or without inhibitors or antibody. A, after
48 h, cells were harvested and counted. B, after
24 h, cells were fixed and the proportion of apoptotic nuclei
determined after Hoechst staining. The following concentrations were
used: 2 µM C2, 10 nM K-252a, 10 µg/ml
anti-p75NTR-blocking antibody (Euromedex), 10 µM PD98059, 18 µM SN50. For A
and B, results are expressed as the means + S.D. of five
separate experiments. Significance was determined using the Tukey's
test (*, p < 0.01).
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Fig. 5.
p140trkA and MAPK activation.
MCF-7 cells were treated with 100 ng/ml NGF in the presence or absence
of 10 nM K-252a, 10 µg/ml
anti-p75NTR-blocking antibody, or 10 µM
PD98059. p140trkA (A) and MAPK activation
(B) were determined after immunoprecipitation using
polyclonal anti-TrkA and monoclonal anti-ERK2 antibodies, respectively.
After SDS-PAGE and electroblotting, nitrocellulose membranes were
counterprobed with the PY20 anti-phosphotyrosine antibody. For
detection of TrkA (A) and MAPK (B) activation,
the lower panel shows reprobing of the blots with the
immunoprecipitating antibody.
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Fig. 6.
Effect of different neurotrophins on MCF-7
cells growth and survival. MCF-7 cells were serum-deprived in
minimum essential medium, and after 24 h the neurotrophins (100 ng/ml NGF, 50 ng/ml BDNF, 50 ng/ml NT-3, 100 ng/ml NT-4/5) were added.
A, after 48 h, cells were harvested and counted. In
contrast with NGF, neither BDNF, NT-3, nor NT-4/5 displayed significant
bioactivity (for concentrations up to 400 ng/ml). B, MCF-7
cells were serum-deprived in minimum essential medium and treated with
2 µM C2, with or without neurotrophins (100 ng/ml NGF, 50 ng/ml BDNF, 50 ng/ml NT-3, 100 ng/ml NT-4/5). After 24 h, cells
were fixed and apoptotic nuclei percentage was determined after Hoechst
staining under an Olympus-BH2 fluorescence microscope. For measurement
of both cell number and apoptosis, results are expressed as the
means ± S.D. of five separate experiments. Significance was
determined using the Tukey's test (*, p < 0.01).
C, TrkB and TrkC mRNA expression in MCF-7 cells. Agarose
gel electrophoresis of RT-PCR products reveals TrkA expression, but no
TrkB or TrkC expression in MCF-7 breast cancer cells. Human NT2 cells
were used as positive control for the expression of TrkB and TrkC.
Lane 1, NT2-negative control without RT step; lane
2, NT2-positive control; lane 3, MCF-7 cells-negative
control without RT step; lane 4, MCF-7 cells.
B Involvement in the Anti-apoptotic Effect of NGF--
The
inhibitory effect of SN50 on the NGF anti-apoptotic activity indicated
the potential involvement of NF-
B in the signaling leading to the
protective activity of this growth factor. To further investigate this
phenomenon, we studied the effect of NGF on the nuclear translocation
of NF-
B, as well as the consequence of transfection by IkBm (an
inhibitor of NF-
B) or by c-rel and rel-A (constitutively active
subunits of NF-
B) on the NGF-mediated anti-apoptotic activity in
MCF-7 cells. Western blotting revealed no change in the nuclear levels
of NF-
B (p65) during apoptosis induced by C2 (Fig.
7). In contrast, the addition of NGF on
C2-treated cells induced a translocation of NF-
B from cytoplasm to
nucleus. Computerized quantification revealed a doubling p65 band
intensity normalized to the total intensity of the lane (data not
shown). Moreover, this NF-
B nuclear translocation was inhibited by
the presence of p75NTR-blocking antibody or SN50, but was
not affected by K-252a and PD98059. Interestingly, in the absence of
C2-induced apoptosis NGF was not able to induce the nuclear
translocation of NF-
B, confirming previous observations that
p75NTR-mediated NF-
B activation requires cell stress
(25). Transfection of MCF-7 cells with IkBm, an inhibitor of NF-
B,
reversed the anti-apoptotic effect of NGF (Fig.
8A). As a control, we
transfected MCF-7 cells with an empty vector; no effect was observed.
In addition, transfection with activators of the NF-
B pathway, c-rel
or rel-A (Fig. 8B), resulted in an inhibition of C2-induced
apoptosis of MCF-7 cells, even in absence of NGF, confirming the
involvement of NF-
B family members in human breast cancer cell
survival.
View larger version (58K):
[in a new window]
Fig. 7.
Activation of NF- B
during NGF anti-apoptotic effect. MCF-7 cells were treated with
100 ng/ml NGF in the presence or absence of 10 nM K-252a,
10 µM PD98059, or 10 µg/ml anti-p75NTR
blocking antibody (Euromedex). Proteins were detected after SDS-PAGE of
nuclear extract preparations and immunoblotting with rabbit
anti-NF-
B p65. The lower panel shows immunoblotting with
anti-lamin B.
View larger version (22K):
[in a new window]
Fig. 8.
Modulation of NGF anti-apoptotic effect by
I Bm, c-rel, and rel-A transfection. MCF-7
cells were co-transfected with EGFPC1 and either I
Bm (A)
or c-rel or rel-A (B) using the Lipofectin reagent. Controls
were performed with both PCDNA3 and EGFPC1 (for I
Bm experiments)
and both PSVK3 and EGFPC1 (for c-rel and rel-A
experiments). After 24 h, cells were serum-deprived in minimum
essential medium and either C2 or neurotrophins added for another 24-h
period. Cells were then fixed and the percentage of apoptotic nuclei in
transfected cells (with the expression of GFP as a transfection
control) determined after Hoechst staining. Results are expressed as
the means ± S.D. of six separate experiments. Significance was
determined using the Tukey's test (*, p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or tumor necrosis factor-
can inhibit
growth and induce apoptosis in these cells (27). Recently we have shown
that NGF, which was primarily described for its neurotrophic
properties, is a strong mitogen for cancerous but not for normal human
breast epithelial cells, suggesting a crucial function for this factor
in the initiation and progression of human breast tumors (13). In the
present study, we have shown that the breast cancer cells express
transcripts for both TrkA and p75NTR receptors. In
contrast, no expression of TrkB and TrkC was found in any of the breast
cancer cells tested, in accordance with the fact that BDNF, NT-3, or
NT-4/5 have no mitogenic effect for these cells. The presence of NGF
receptors has been detected previously in breast cancer cells (28), and
low levels of NGF receptor expression have recently been reported in
other breast cancer cell lines (29), leading to the hypothesis of a
recruitment and cooperation between p140trkA and
p185Her-2 for the induction of mitogenesis by NGF. Our
results indicate a stimulation of p140trkA tyrosine kinase
activity and of the MAPK cascade by NGF, and the use of the
pharmacological inhibitors K-252a and PD98059 demonstrate the
requirement for these signals in NGF-induced MCF-7 cell proliferation. The induction of MAPK activity required p140trkA activation,
but p75NTR did not appear to be involved, because
p75NTR-blocking antibodies did not have any effect on
NGF-induced MAPK activation and cell proliferation. In contrast,
p75NTR-blocking antibodies exhibited an inhibition of
NGF-induced survival, attesting to the functionality of these blocking
antibodies. Thus, the mitogenic activity of NGF requires the
p140trkA and MAPK cascade independently of the
p75NTR receptor. This signaling pathway for the
NGF-proliferative effect appears to be similar to that which is
described for the neurotrophic activity of this factor. For example, in
PC-12 pheochromocytoma cells, disruption of p75NTR does not
result in an inhibition of the NGF differentiative activity, which is
mediated by the p140trkA/MAPK pathway (30). Interestingly, it
has also been shown in PC-12 cells that NGF induces survival and
differentiation through two distinct signaling pathways, because the
activation of the MAPK cascade is required for the differentiative but
not the protective activity of NGF (31). These data emphasize the
similarities between the mitogenic and neurotrophic signaling pathways
of NGF.
B) and the regulation of
cell survival/apoptosis. In some cases apoptosis was shown to develop
following NGF binding to p75NTR, although in other cases it
appeared to occur in the absence of ligand (spontaneous apoptosis) and
was reversed by NGF (35). The C2 reagent used here is known to induce
apoptosis in breast cancer cells such as MCF-7 (21, 22). Morphological
analysis after Hoechst staining and the inhibition of PARP cleavage
demonstrated that NGF rescues breast cancer cells from C2-induced cell
death. Interestingly, K-252a and PD98059 did not affect the
anti-apoptotic activity of NGF, indicating that p140trkA
tyrosine kinase and MAPK activities are not necessary for the protective effect. Previous reports have noted that NGF is able to
elicit its biological effects through p75NTR receptors and
independently of p140trkA in neurons (36, 37) and Schwann cells
(38). In our experiments, a specific role for p75NTR in the
cell survival effect was first suggested by the fact that other
neurotrophins (interacting with p75NTR and not with
p140trkA) are also able to protect cells from death while
having no impact on cell proliferation. The crucial role of
p75NTR was further demonstrated by the use of
p75NTR-blocking antibodies, which completely reversed the
protective effect of NGF from C2-induced apoptosis. Moreover, BDNF,
NT-3, and NT-4/5, all of which can bind P75NTR, can also
stimulate breast cancer cell survival. Because TrkB and TrkC are not
expressed in breast cancer cells, these data emphasize the role played
by p75NTR in the anti-apoptotic effect of NGF. Activation
of p75NTR specifically induces NF-
B independent of
p140trkA in several cell types, including Schwann cells (38).
To explore the involvement of NF-
B in the NGF survival effect, we
first tested SN50, which inhibits the nuclear translocation of this transcription factor (39). We found that it blocked the anti-apoptotic effect of NGF without affecting the p140trkA/MAPK cascade or
cellular proliferation. The involvement of NF-
B was further
demonstrated by transfection with a mutated form of IkBa, which blocked
NF-
B translocation to the nucleus. MCF-7 cells transfected by
mutated IkBa were not rescued from C2-induced apoptosis by NGF,
confirming the involvement of NF-
B in the anti-apoptotic activity
mediated by p75NTR. Similar observations have been made in
PC12 cells in which the blocking of p75NTR-mediated
activation of NF-
B resulted in an enhancement of apoptosis (40). In
addition, transfections by c-rel or rel-A, which are constitutively
activated forms of NF-
B, had a protective effect on MCF-7 cells
treated by C2 in absence of NGF stimulation. c-rel and rel-A belong to
the NF-
B family of transcription factors. The protection from
apoptosis observed after transfection with this factor emphasizes the
role played by NF-
B molecules in the control of breast cancer cell survival.
B
alone. NGF is present in the mammary gland (41, 42) as well as its
transcripts,2 and our present
finding therefore emphasizes that NGF is a crucial regulator of mammary
tumor growth. The inhibition of breast cancer progression through the
targeting p140trkA and p75NTR should be considered
as a potential perspective for the treatment of this pathology.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Rachel Connor (Imperial College, London) and David G. Fernig (University of Liverpool) for critical reading of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Ligue Nationale Contre le Cancer (Comité du Nord) and by the French Ministry of Research and Education.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.
§ Recipients of an Association pour la Recherche sur la Cancer fellowship.
To whom correspondence should be addressed:
EA-1033, batiment SN3, Université des Sciences et Technologies de
Lille, 59655 Villeneuve d'Ascq cedex, France. Tel.:
33-3-20-43-40-97; Fax: 33-3-20-43-40-38; E-mail:
hubert.hondermarck@univ-lille1.fr.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M010499200
2 S. Descamps, R.-A. Toillon, E. Adriaenssens, V. Pawlowski, S. M. Cool, V. Nurcombe, X. L. Bourhis, B. Boilly, J.-P. Peyrat, and H. Hondermarck, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NGF, nerve growth
factor;
PAGE, polyacrylamide gel electrophoresis;
NF-B, nuclear
factor-
B;
BDNF, brain-derived neurotrophic factor;
NT, neurotrophin;
PARP, polyADP-ribose polymerase;
TNF, tumor necrosis factor;
TBP, TATA
box binding protein;
RT-PCR, reverse transcriptase-polymerase chain
reaction;
FCS, fetal calf serum;
DTT, dithiothreitol;
PBS, phosphate-buffered saline;
ERK, extracellular signal-regulated kinase;
GFP, green fluorescence protein;
I
Bm, dominant-negative
I
B
mutant;
bp, base pair(s);
PD98059, Park Davis
98059.
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