From the Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milano, Italy
Received for publication, October 25, 2000, and in revised form, January 11, 2000
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ABSTRACT |
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HER2 oncogene overexpression has been associated
either with proliferation or differentiation and apoptosis. The role of
p53 on these different chances was investigated. Wild type (wt)
p53-IGROV1 cells showed growth inhibition and apoptosis after HER2
transfection, whereas no anti-proliferative effect was observed in its
mutated p53 sub-line unless wt p53 was cotransfected with HER2. Stable HER2 transfectants derived from wt p53 line treated with heregulin- HER2 overexpression is a frequent event in several human cancers,
including breast tumors, and has been correlated with a poor prognosis
(1-4). HER2 is a member of the epidermal growth factor
receptor-related family of receptor tyrosine kinases, which comprises
HER1, HER2, HER3, and HER4 (5, 6). The four proteins are normally
coexpressed in various combinations and can form homo and heterodimers,
which are activated in response to a number of peptide factors (7-9).
The MAPK1 pathway, which has
been implicated in growth and transformation in many cell systems (10,
11), is one of the pathways that may be activated downstream HER2. The
substrates of the MAPK cascade include transcriptional factors such
Jun, Fos, and c-Myc (10), which are thought to activate the cell
cycle machinery and its checkpoints (12).
In vitro studies show that transfection of HER2 in many
cellular models induces proliferation and a malignant phenotype (13, 14). Nevertheless, we recently reported that HER2 transfection resulted
in decreased plating and cloning efficiency, decreased growth rate,
inhibition of entry into the S-phase of the cell cycle, and
differentiation (15), consistent with the induction of differentiation
(16) or/and growth inhibitory effect (17) in some cell lines by
Neu Differentiating Factor (NDF)/heregulin (HRG) and some
antibodies against HER receptors. In breast cancer cells, the mechanism
of growth inhibition and differentiation via HER2 receptor activation
has been suggested to involve p53 (18), and HER2 overexpression in
breast cancer, associated with increased proliferation, is frequently
found in tumors with p53 alterations (19). We therefore tested the
relevance of p53 status for the activity of HER2 in promoting
proliferation using HER2-transfected cells that differ in p53 status.
We show here that p53 status greatly influences the effect of HER2
overexpression in tumor cells in promoting either proliferation or
apoptosis or blocking proliferation.
Cells and Culture Conditions--
The IGROV1 cell line was
originally derived by Dr. Bernard (Institute Gustave Roussy, Villejuf,
France) from a moderately differentiated ovarian carcinoma of an
untreated patient. IGROV1/Pt1, kindly provided by Dr. F. Zunino
(Istituto Nazionale Tumori), was generated by continuous exposure of
IGROV1 cells to increasing concentrations of cisplatin (20).
All cell lines were maintained at 37 °C in a humidified atmosphere
(5% CO2 in air) in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (HyClone) and 2 mM L-glutamine (Sigma). All transfected cells
were cultured as above, except that the culture medium also contained
G-418 (Geneticin) (Life Technologies, Inc.) at 200 µg/ml in the case
of HER2-transfected IGROV1 and IGROV1/Pt1 cells or mock-transfected cells.
Plasmids--
Plasmids pcDNA/HER2 and pEGFP/HER2 were
prepared by excising full-length human HER2 cDNA from the
LTR-2/erbB-2 expression vector (kindly provided by Dr. P. Di Fiore)
(14) by XhoI digestion (Roche Molecular Biochemicals) and
subcloning the fragment into the XhoI restriction site of
plasmid pcDNA1/neo (Invitrogen) in the case of pcDNA/HER2 or
into pEGFP-C2 (CLONTECH) in which the cloned
cDNA was expressed as a fusion product with the N terminus of green
fluorescent protein (GFP) in the case of pEGFP/HER2. Full-length
p53 cDNA, excised by BamHI digestion of plasmid pC53-SN3 (21), was subcloned into plasmid pIREShyg
(CLONTECH) and designated pIRES/p53.
Transfection of HER2 cDNA--
Stable transfections were
conducted with pcDNA/HER2, and transient transfections were
conducted with pEGFP/HER2. For stable transfections, cells at 80%
confluence in serum-free medium were transfected with 30 µg of
Lipofectin (Life Technologies, Inc.) for selection control or with 30 µg of Lipofectin plus 3 µg of plasmid. Cells were left at 37 °C
for 5 h and, after replacement of serum-free with complete medium,
maintained for an additional 48 h. Cells were trypsinized and
plated in the presence of 400 µg/ml G-418 until all nontransfected
cells were dead. Individual G-418-resistant colonies were picked,
expanded, and maintained in the presence of G-418 (200 µg/ml).
Control cells were transfected with pcDNA1/neo only ("mock").
Individual colonies as well as bulk cultures of G-418-selected cells
were tested for p185HER2 surface expression by FACScan analysis.
For transient transfection, the same Lipofectin/DNA ratio as for stable
transfections was maintained; cells were seeded in duplicate chamber
slides (Lab-Tek) and, 24 h later, washed with phosphate-buffered
saline (PBS) and fixed with 2% paraformaldehyde. After a wash with
PBS, nuclei were stained with 25 µg/ml Hoechst reagent
(4,6-diamidino-2-phenylindole) for 30 min at room temperature in the
dark. Either the percentage of transfection or apoptosis was evaluated
by visual count under a fluorescence microscopy.
Cotransfection of HER2 and wt p53--
Cells were seeded in
duplicate chamber slides (Lab-Tek) and, at 80% confluence, transiently
cotransfected. Each well received 0.2 µg of each plasmid and 2 of
µg of Lipofectin in serum-free medium. The plasmid combinations used
were empty pEGFP-C2 and pIREShyg, pIRES/p53 and empty pEGFP-C2,
pEGFP/HER2 and empty pIREShyg, and pIRES/p53 and pEGFP/HER2. Cells were
kept at 37 °C for 5 h and, after replacing serum-free with
complete medium, maintained in culture for 24 h and treated as
described for transient transfections.
Colony Assays--
Cells were transfected with either
pcDNA1/neo or pcDNA/HER2 (same Lipofectin/DNA ratio as for
stable tranfection) and plated in duplicate 6-well plates (5 × 105 cells/well). After 48 h, medium was replaced with
fresh medium containing 300-800 µg/ml G-418, depending on the cell
line. After 3 weeks of selection, plates were stained with TB
methylene blue (Difco) for 15 min. Colonies were counted, and the
percentage of control colony formation was calculated.
Flow Cytometric Analysis--
Trypsin-detached cells were
incubated for 45 min at 0 °C with 10 µg/ml
anti-p185HER2 monoclonal antibody (mAb) MGR6 (22) in PBS
containing 0.03% bovine serum albumin. After several washes, cells
were further incubated for 45 min at 0 °C with fluorescein-linked
goat anti-mouse antibody (Kierkegaard and Perry Laboratories). After
washing, cells were assessed for fluorescence using a FACScan flow
cytometer with LYSIS TM II software (Becton-Dickinson).
Proliferation Assay Sulforhodamine B (SRB)--
Cells were
seeded at 10 × 103 cells/well in 96-well plates in
200 µl of culture medium and grown for 1, 2, 3, and 6 days. Each test
was performed in six replicates. Briefly, cells were fixed by
incubation with 10% trichloroacetic acid at 4 °C for 1 h
followed by 5 washes with distilled water. Cells were stained by the
addition of 1% acetic acid, 0.4% (w/v) SRB (Sigma) solution to the
culture medium at room temperature; after 30 min, plates were washed
with 1% acetic acid and air-dried. After the addition of 10 mM Tris-HCl, pH 10.5, to dissolve the SRB bound to cellular
proteins, absorbance at 550 nm, proportional to the number of cells
attached to the culture plate, was measured by spectrophotometry.
[3H]Thymidine Incorporation--
Cells were seeded
at 10 × 103/well in 96-well plates in complete medium
for 3 h. Cells were then serum-starved for 48 h and treated
with 10 ng/ml HRG Soft Agar Assay--
Cells (5 × 103/well) were
seeded in 6-well plates in semisolid medium containing 0.3% Bacto-Agar
(Difco) supplemented with 30% fetal calf serum and 300 µg/ml G-418
over a 0.6% agarose layer. Colonies were scored after a 3-week
incubation at 37 °C in 5% CO2 in air.
Cell Cycle Analysis--
Cells were cultured for 48 h in
serum-free medium and treated with 10 ng/ml HRG Western Blot Analysis--
Cells were scraped and lysed in hot
SDS buffer (0.125 nM Tris-HCl, pH 7.4, 5% SDS, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4). Cell lysates were
cleared by centrifugation. Protein concentration was determined by the
BCA protein assay reagent (Pierce). For each sample, 50-100 µg of
total protein extract was fractionated by SDS-polyacrylamide gel
electrophoresis as described (23) and blotted onto a nitrocellulose
membrane (Hybond C, Amersham Pharmacia Biotech). Membranes were
incubated with the primary antibody for 2 h followed by incubation
with horseradish peroxidase-linked sheep anti-mouse Ig (1:5000,
Amersham Pharmacia Biotech) and visualized using the ECL detection
system (Amersham Pharmacia Biotech) according to the supplier's instructions.
For MAPK studies, cells were cultured for 48 h in fetal calf
serum-free medium and treated with 10 ng/ml of HRG
mAbs used were anti-p185HER2 Ab-3 (1 µg/ml; Oncogene
Science), anti-phosphotyrosine 4G10 (4 µg/ml; Upstate Biotechnology,
Inc.), p53-DO7 (1:100, Novocastra Laboratories Ltd.), anti-MDM2
Ab-1 (1 µg/ml, Calbiochem), c-Myc Ab-5 (1 µg/ml Neo Markers),
p44/42 MAPK antibody (1:1000; BioLabs Inc.), Phospho-p44/42MAP
kinase (Thr-202/Thr-204) antibody (1:1000, BioLabs Inc.), and
anti-actin clone AC40 (1:500, Sigma).
Northern Blot Analysis--
RNA was extracted using the Talent
RNA extraction kit (Talent s.r.l.), according to the
manufacturer's instructions. RNA (10 µg/sample) was electrophoresed
on a 1% agarose-formaldehyde gel, transferred to nitrocellulose
filters (Amersham Pharmacia Biotech), and immobilized by
UV-cross-linking. Hybridization was carried out using a
[32P]dCTP (Amersham Pharmacia Biotech) random-primed
(Roche Molecular Biochemicals) probe comprising full-length p53
cDNA, obtained by BamHI digestion of pC53-SN3 (21).
After stripping, membranes were hybridized with a control
[32P]dCTP IGROV1 ovary carcinoma cells, which carry wt p53, and the
cisplatin-resistant variant IGROV1/Pt1, carrying a p53 mutated at codons 270 and 282, were transfected with a HER2 expression vector. Colony assay revealed dramatic inhibition in the growth of IGROV1 transfectants as compared with mock-transfected cells (Fig.
1A), whereas the clonogenic
potential of the mutant p53 IGROV1/Pt1 transfectants was comparable
with that of mock controls.
1 or epidermal growth factor showed a decrease in proliferation due to a
G2/M cell cycle block despite normal
mitogen-activated protein kinase activation. In these HER2
transfectants, c-Myc and p53 expression were increased, whereas MDM2
was dramatically down-modulated. By contrast, growth factors
stimulation of HER2 transfectants with mutated-p53 induced progression
through the cell cycle. Together, our data point to a regulatory role
for p53 in HER2 signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (NeoMarkers) or 20 ng/ml EGF (Life Technologies,
Inc.) for 24 h and with 1 µCi/well
[methyl-3H]thymidine (Amersham Pharmacia
Biotech), 1 mCi/ml for the last 4 h. Cells were washed with
ice-cold PBS, precipitated with 10% trichloroacetic acid, and
solubilized in 100 µl of 0.2 N NaOH, 1% SDS. Lysates
were neutralized with 100 µl of 0.2 N HCl, and incorporated radioactivity was quantitated by scintillation counting.
1 or 20 ng/ml EGF for
24 h at 37 °C or left untreated. Cells were harvested, fixed in
0.5% paraformaldehyde for 5 min, and permeabilized in 0.1% Triton
X-100. Cells were then incubated at 37 °C for 30 min in the presence
of RNase A (1 mg/ml; Sigma) and stained for 30 min at 0 °C with
propidium iodide (50 µg/ml) in PBS containing 0.03% bovine serum
albumin. Fluorescence was measured using a FACScan flow cytometer with LYSIS TM II software, and data were analyzed with CellFIT software (all
Becton Dickinson).
1 or 20 ng/ml EGF
for 10 min at 37 °C. After treatment, cells were lysed for 45 min on
ice in 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4. Cell lysates were cleared by centrifugation. Total lysate (50 µg) was resolved in a
12.5% polyacrylamide gel and blotted as described above.
-actin probe (24). Densitometric analysis
was performed by PhosphorImager scanning using the ImageQuant System
(Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, colony formation of
HER2-transfected cells bearing wt or mutated p53. Cells were
transfected with pcDNA1/neo (control) or pcDNA1/HER2. After 3 weeks of selection, colonies were counted. Results are given as mean
percentage (bars, S.E. of two separate experiments) of
control colony formation. B, apoptosis in HER2-transfected
cells differing in p53 status. Cells were seeded in chamber slides and
transiently transfected with pEGFP/HER2 in which the cloned HER2
cDNA was expressed as a fusion product with the N terminus of GFP
or with empty pEGFP-C2. Apoptosis is expressed in mean percentage of
apoptotic nuclei relative to the total number of green-labeled cells
counted (bars, S.E. of 4-5 experiments). C,
IGROV1/Pt1 cells were transiently cotransfected with pIRES/p53, which
contains full-length wt p53 cDNA, and pEGFP/HER2, in which the
cloned HER2 cDNA was expressed as fusion with the N terminus of
GFP. Mean percentages of apoptotic nuclei were evaluated
(bars, S.D. of two replicates). 1, empty pEGFP-C2
and pIREShyg; 2, pIRES/p53 and empty pEGFP-C2; 3,
pEGFP/HER2 and empty pIREShyg; 4, pIRES/p53 and
pEGFP/HER2.
To determine whether the reduced colony formation in HER-2-transfected wt p53-bearing cells reflected apoptosis, IGROV1 and IGROV1/Pt1 cells were transiently transfected with an expression vector in which HER2 cDNA was fused to the N terminus of GFP and the number of 4,6-diamidino-2-phenylindole-stained apoptotic nuclei in the green-fluorescing cells was evaluated (Fig. 1B). HER2-transfected IGROV1 cells underwent a significantly higher rate of apoptosis than did the mock-transfected cells (p = 0.009); indeed, 31% of green-stained HER2-transfected IGROV1 cells were apoptotic versus 11% of green-stained GFP-transfected cells. By contrast, the percentage of IGROV1/Pt1 apoptotic cells was similar (10-11%) after either HER2 or mock transfection. In HER2-transfected IGROV1/Pt1 cells transiently cotransfected with wt p53 cDNA, the apoptotic rate was significantly (p = 0.025) increased (32%) as compared with the rate with single vectors alone (Fig. 1C). Together these data indicate that HER2 ectopic expression induces apoptosis only in cells with wt p53.
The in vitro growth properties of stable transfectants
derived from IGROV1 and IGROV1/Pt1 cells were evaluated. FACScan
analysis revealed comparable levels of HER2 expression in two IGROV1
clones (#5, #11) and two IGROV1/Pt1 clones (#24, #35), randomly
selected among those with high HER2 expression, (Fig.
2A). Note that nontransfected IGROV1/Pt1 cells spontaneously displayed a higher membrane HER2 level
than did IGROV1 cells.
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Both the IGROV1/HER2 clones displayed a lower proliferation rate, as evaluated by SRB analysis (Fig. 2B, left) and [3H]thymidine incorporation (Fig. 2B, right), than mock-transfected cells. By contrast, HER2 expression conferred an evident growth advantage to IGROV1/Pt1 cells as compared with mock transfectants. In an anchorage-independent colony-forming assay, the capacity of HER2-transfected IGROV1 cells to form clones in soft agar was dramatically diminished (90% reduction).
To determine whether the mitogenic pathway primed by HER2 stimulation
was activated, transfectants were treated with HRG1 or EGF, and cell
lysates were probed for MAPK activation (Fig. 3A). The addition of either
ligand to IGROV1/HER2 #5 induced a higher activation (2- and 2.8-fold
with HRG
1 and EGF, respectively) of MAPK than in mock-transfected
cells (1.4- and 1.9-fold). In IGROV1/Pt1 transfectants, either
treatment activated both mock (2- and 3-fold) and IGROV1/Pt1/HER2 #24
transfectants (2.5- and 3.5-fold).
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Analysis of cell cycle progression in HER2- or mock-transfected cells
after treatment with HRG1 or EGF revealed an increase in the
percentage of S phase cells and a corresponding decrease in
G2/M phase cells, except in IGROV1/HER2 #11, where both
treatments induced a decrease in S phase and an increase in
G2/M phase cells (Fig. 3B).
Western analysis indicated a dramatic increase in p53 levels in both
IGROV1/HER2 clones as compared with the mock transfectants, whereas in
IGROV1/Pt1 cells, in which a high level of p53 was already present
before HER2 transfection, no difference in p53 levels was
detected after transfection (Fig.
4A).
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Northern analysis indicated that p53 mRNA levels of IGROV1/HER2 #5
normalized to -actin were 5 times higher than those of IGROV1/mock
cells, whereas p53 mRNA levels of IGROV1/Pt1 transfectants were
almost the same as in the mock controls and comparable to levels in
IGROV1/HER2 cells (Fig. 4B). Western analysis indicated a
dramatic down-modulation of MDM2 protein in IGROV1/HER2 #5 cells as
compared with IGROV1/mock; in IGROV1/Pt1 cells, MDM2 levels were very
low in both mock and HER2 transfectants. The relative p53 protein
levels are shown to emphasize that low MDM2 expression is associated
with high p53 expression (Fig. 4C). On the other side, HER2
transfection led to increase in c-Myc protein levels in IGROV1 cells;
conversely, this was not observed in IGROV1/Pt1 cells (Fig.
4D).
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DISCUSSION |
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In this paper, we demonstrate that p53 status greatly influences the final effect of HER2 overexpression in tumor cells in promoting proliferation or apoptosis and a block in proliferation. Indeed, HER2 overexpression was associated with proliferation only in tumor cells with mutated p53. By contrast, HER2-transfected cells bearing wt p53 became apoptotic shortly after transfection, and after stabilization of apoptosis-resistant cells, showed decreased proliferation. These data are consistent with a p53-dependent block of the cells in response to an excess of HER2 signaling induced by transfection.
The treatment with HRG1 or EGF induced MAPK activation in the
HER2-transfected lines, indicating that the transfected HER2 was
functional and the mitogenic pathway mediated by HER2 was primed, but
some molecules downstream of MAPK shift the signal from induction to
inhibition of proliferation. Although MAPK activation is usually
correlated with proliferation, HER2-transfected cells expressing wt p53
and treated with HRG
1 or EGF, which activated MAPK, led to decreased
cell proliferation. Apparently, cells were blocked in G2/M
phase of the cell cycle.
HER2-transfected IGROV1 cells expressing wt p53 showed increased p53 mRNA and protein. The increased p53 protein expression was not due to a mutation acquired during culture leading to lower turnover (25). Indeed, IGROV1/HER2 cells still underwent apoptosis after treatment with a p53-dependent apoptotic agent such as mitomycin C, whereas IGROV1/Pt1 cells did not (data not shown). MDM2 is an important component of the p53 degradation pathway and is a target for transcriptional transactivation by p53 (26). In normal cells, low levels of inactive p53 protein are maintained by the MDM2 protein, which blocks p53 transcriptional activity in the nucleus and shuttles p53 into the cytoplasm for degradation by the cytoplasmic proteasome (27). In tumor cells, mutations of p53 abrogate not only the tumor suppressor activity but also the ability to activate MDM2 expression. As a result, mutated p53 proteins are unusually stable and accumulate in tumor cells (25). In keeping, accumulation of mutated p53 in both HER2- and mock-transfected IGROV1/Pt1 cells corresponded to nearly undetectable levels of MDM2. P53 protein levels rise in response to many stimuli such as stress or DNA damage, and p53 activation induces a cell cycle block or apoptosis (28, 29). In HER2-transfected IGROV1 cells, where p53 expression was dramatically increased, MDM2 protein levels were nearly undetectable. The observed G2/M cell cycle block in IGROV1/HER2 cells is consistent with recent studies suggesting that p53 regulates the G2 checkpoint in the cell cycle (30). In cells with wt p53 and aberrant HER2 signaling, p53 might act as a positive control and block the cells in G2/M phase. In cells with mutated p53, such control on HER2 is absent, followed by proliferation in response to excess HER2 signaling.
P53 levels are also indirectly regulated by p14Arf, which binds to MDM2 protein, sequesters it, and consequently induces its degradation (31). p14Arf is transcriptionally regulated by c-Myc (27). Furthermore c-Myc has recently been proposed as a primary effector of HER2-mediated activity (32). Thus it is possible that the signaling pathway of HER2 is regulated by p53 via c-Myc. In keeping, c-Myc levels were found increased in HER2-transfected IGROV1 cells. Concerning IGROV1/Pt1 cells both mock- and HER2-transfected cells, c-Myc levels were high, consistent with high HER2 expression levels in IGROV1/Pt1 mock cells already present before HER2 transfection. The presence of mutated p53 in IGROV1/Pt1 might determine the positive selection of HER2-expressing cells in which the proliferative effect of this oncoprotein is not contrasted by p53.
The control of HER2 signaling by p53 provides an explanation for the
association between HER2 overexpression and alteration of p53 in breast
carcinomas in vivo (19) and could have implications for
treatment with herceptin, since treatment should be more effective in
tumors with mutated p53. Together, our data point to the importance of
p53 regulation in the HER2-signaling pathway and suggest that the
repair of p53 function might counter the tumor aggressiveness associated with HER2 overexpression.
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ACKNOWLEDGEMENTS |
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We thank L. Mameli for preparing the manuscript and M. Azzini for photographic reproduction.
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FOOTNOTES |
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* This work was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC).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.
To whom correspondence should be addressed: Molecular Targeting
Unit, Dept. of Experimental Oncology, Istituto Nazionale Tumori, Via
Venezian 1, 20133 Milano, Italy. Tel.: 39-02-2390571; Fax: 39-02-2362692; E-mail: menard@istitutotumori.mi.it.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M009732200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; HRG, heregulin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; wt, wild type; mAb, monoclonal antibody; SRB, sulforhodamine B; EGF, epidermal growth factor.
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