(CANCER RESEARCH 51, 1898-1903. April I, 1991]
Overexpression of Epidermal Growth Factor and Insulin-like Growth Factor-I
Receptors and Autocrine Stimulation in Human Esophageal Carcinoma Cells1
Shan-Chun Chen, Chen-Kung Chou, Fen-Hwa Wong, Chungming Chang, and Cheng-po Hu2
(jraduale Institute of Microbiology and immunology, National Yang-Ming Medical College ¡S-C.C., C-K. C., F-H. W., C. C., C-p. HJ; Department of Medical
Research, Veterans General Hospital (C-K. C., C. C., C-p. H.J; and Institute of BiomédicalSciences, Academia Sinica [S-C. C./, Taipei, Taiwan, Republic of China
ABSTRACT
cells overexpress the EGF receptor (2, 5, 6). However, the
growth regulation of esophageal carcinoma cells by EGF or by
other growth factors remains to be examined. In the present
study, we report that EGF, TGF-a, and IGF-I are potent
mitogens for a receptor-overexpressed human esophageal car
cinoma cell line, CE48T/VGH, under serum-free conditions.
Autocrine growth regulation by TGF-a and IGF-I in CE48T/
VGH cells is also supported by this study.
The growth-stimulatory effects of epidermal growth factor (EGF),
transforming growth factor a (TGF-a), and insulin-like growth factor-I
(IGF-I) on the human esophageal carcinoma cell line CE48T/VGH were
evaluated. Under serum-free conditions, EGF, TGF-a, and IGF-I pro
moted 3.6- to 4.1-fold increased cell proliferation. Scatchard analyses
and Northern blot hybridization revealed that both the EGF/TGF-a
receptor and the IGF-I receptor were overexpressed in CE48T/VGH
cells. Furthermore, ligand-dependent autophosphorylation of the EGF
receptor and the IGF-I receptor was clearly detected using antireceptor
MATERIALS AND METHODS
and antiphosphotyrosine antibodies. Autocrine regulation was strongly
indicated by the following evidence: (a) CE48T/VGH cells were found to
Materials. Mouse EGF (culture grade) and recombinant human
express TGF-a and IGF-I genes, (b) serum-free conditioned medium TGF-a were purchased from Collaborative Research (Waltham, MA).
promoted the growth of CE48T/VGH cells and stimulated the autophos
Recombinant IGF-I was purchased from Bachern (Terranee, CA).
phorylation of the EGF/TGF-a receptor and the IGF-I receptor, and (<•)
Antiphosphotyrosine antibody was from Amersham (England). Mono
the addition of IGF-I receptor antibodies significantly suppressed CE48T/
clonal antibody against EGF receptor was obtained from Oncogene
VGH cell growth under serum-free conditions. Our studies suggest that
Science, Inc. (Mineóla, NY). Monoclonal antibody against IGF-I re
the overexpression of EGF and IGF-I receptors and autocrine growth
ceptor (aIR-3) was generously provided by Dr. Fred Kull (Division of
regulation may concertedly control the proliferation of esophageal carci
Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC).
noma cells.
Radioactive
chemicals,
including
Na'"I,
'"I-EGF,
p'S)methionine, [a-"P]dCTP, and [r-12P)ATP, were from New England
Nuclear (Boston, MA). IGF-I was iodinated by the chloramine T
INTRODUCTION
method. DMEM and Ham's F-12 medium were from GIBCO (Grand
The growth and differentiation of normal and transformed
cells are controlled by numerous growth factors. The binding
of growth factors to their specific receptors on the cell surface
may stimulate the receptor-associated tyrosine kinases, which
transmit mitogenic signals, and lead to cell proliferation. Neoplastic cells are often found to have altered synthesis of recep
tors for growth factors. Amplification and overexpression of
the EGF3 receptor gene have been observed in human carci
noma cells (1-6) and sometimes correlated with the invasive
and metastatic properties of the tumor cells (3). Furthermore,
EGF can stimulate anchorage-independent
growth of carci
noma cells with an amplified and overexpressed EGF receptor
gene (4). Thus, a close relationship has been suggested between
the activation of the EGF receptor gene and carcinogenesis.
Additional evidence relating growth factors to oncogenesis
comes from the observation that many transformed cells pro
duce growth factors that many stimulate their own growth in
an autocrine manner (6-11). This observation suggests that the
ability of a cell to produce a factor(s) which can support its own
growth might be a general mechanism contributing to the
unlimited growth capacity of tumor cells.
Recent studies have shown that human esophageal carcinoma
Received 9/4/90; accepted 1/21/91.
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.
' This work was supported by Grant NSC79-0412-B075-37 from the National
Science Council, Republic of China.
2 To whom requests for reprints should be addressed.
' The abbreviations used are: EGF, epidermal growth factor: PCS, fetal calf
serum: PMSF. phenylmethylsulfonyl fluoride; HEPES, 4-<2-hydroxyethyl)-lpiperazineethanesulfonic acid: HBSS, Hanks' balanced salt solution; SDS, so
dium dodecyl sulfate; cDNA, complementary DNA; PAGE, polyacrylamide gel
electrophoresis; BSA, bovine serum albumin; IGF-1, insulin-like growth factor I;
TGF-n, transforming growth factor «;SSC, standard saline citrate; DM EM,
Dulbecco's modified Eagle's medium; SFCM, serum-free conditioned medium.
Island, NY). Protease inhibitors aprotinin, antipain, pepstatin A, and
PMSF were from Sigma Chemical Co. (St. Louis, MO).
Cell Culture. CE48T/VGH cells (12) were maintained in a 2:1 (v:v)
mixture of Ham's F-12 medium and DMEM (F2D1), supplemented
with 5% FCS, 100 If/ml penicillin, 100 ¿ig/mlstreptomycin, 250 n%/
ml fungizone, 2 HIML-glutamine, and 1 MMnonessential amino acids
and buffered with 40 HIM sodium bicarbonate and 25 mM HEPES.
Human lung carcinoma cell line CaLu-1 (13), human hepatoma cell
line HA22T/VGH (14), human epidermoid carcinoma cell line A431
(15), and normal human fibroblast line WI-38 (16) were maintained in
DMEM containing 10% FCS (complete medium). Cells were cultured
at 37°Cin a humidified atmosphere containing 5% CO2. Subculture of
cells was performed every 3 to 4 days after the culture had reached
confluency, with 0.05% trypsin plus 2 mM EDTA.
Cell Proliferation Assays. CE48T/VGH cells were seeded in 24-well
plates, at a density of 5 x IO4cells/well, with F2D1 medium containing
5% FCS. After 18 h, cells were placed in serum-free (SF-F2D1 ) medium
for another 24 h. Triplicate wells were then fed with growth factorcontaining SF-F2D1 medium. The media were refreshed once after 2
days. Viable cell numbers were determined 4 days after growth factor
treatment, by trypan blue exclusion.
For proliferation-inhibition studies, various amounts of anti-IGF-I
receptor antibody or control antibody were added into SF-F2D1 me
dium 24 h after serum starvation. The antibody-containing media were
refreshed once after 2 days. Viable cell numbers were determined 4
days after the addition of antibodies.
Collection of SFCM. CE48T/VGH cells were plated in 150-mm Petri
dishes with F2D1 medium containing 5% FCS. At 90% confluency,
the medium was replaced by SF-F2D1 medium and refreshed 1 day
later. The conditioned media were collected every 2 days thereafter,
pooled, concentrated 100-fold by polyethylene glycol 10,000, dialyzed
against fresh SF-F2D1 medium, and stored at -20°C before use.
I25l-Ligand Binding Assays. CE48T/VGH cells were seeded in 24well plates, at a concentration of 1.5 x IO5cells/well, and cultured for
2 days in F2D1 medium containing 5% FCS. The cells were washed
twice with HBSS and incubated at room temperature for 90 min with
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EGF AND IGF-I RECEPTORS AND AUTOCRINE STIMULATION
1 ng/ml '"¡-labeled ligands in binding buffer (HBSS with 20 mM
HEPES, 0.5% BSA, and various concentrations of unlabeled ligands,
ranging from 0.5 to 100 ng/ml, at pH 7.6). The cells were washed 5
times with ice-cold phosphate-buffered saline and lysed by 0.2% SDS.
The radioactivity was determined with a gamma-counter. The numbers
of surface receptors and the affinity constants were calculated by
Scatchard analysis. Nonspecific binding was determined in incubations
containing 200 ng to 1 ne unlabeled ligands.
RNA Isolation and Northern Blot Analysis. Total RNA was isolated
by guanidine isothiocyanate solubilization and centrifugation through
a CsCl cushion. Polyadenylated RNA was enriched by oligothymidylate-cellulose chromatography. electrophoresed on 1% agarose gels
containing 2.2 M formaldehyde, and transferred to Hybond nitrocellu
lose filters (Amersham, UK) in 20x SSC (3 M NaCl, 0.3 M trisodium
citrate). The RNA blots were hybridized with ';P-labeled probes for 20
h at 42°C,in a solution containing 5x SSC, 50% formamide, 5x
Denhardt's reagent, and 0.1% SDS. The filters were washed twice with
0.2x SSC/0.1% SDS, at 55°Cand at 60°Cfor 40 min, and once with
0.1 x SSC/0.1 % SDS at 60°Cfor 40 min, followed by autoradiography.
The following DNA fragments were used as probes: a 768-base pair
£coRIfragment of human EGF receptor cDNA (17), plasmid pIGF-IR.76 containing a portion of human IGF-I receptor cDNA (18), a 727base pair Pstl fragment of human IGF-I cDNA (19), a 1.4-kilobase
EcoRI fragment of human TGF-a cDNA (20), and a 1.9-kilobase
fragment of human EGF cDNA from phEGFIS (21).
Preparation of Membrane Fractions. CE48T/VGH cells were grown
in Petri dishes ( 150-mm diameter) until 80% confluent. The monolayers
were washed 3 times with ice-cold Ca:+/Mg2*-free HBSS and scraped
off into lysing buffer (0.1 M sodium phosphate, 5 mivi EDTA, 250 mM
sucrose, 10 Mg/ml aprotinin, 20 ¿ig/mlantipain, 20 Mg/ml pepstatin A,
2 mM PMSF, pH 7.4), at 5 x IO6 cells/ml. All the subsequent steps
were carried out at 4°C.Cells were disrupted by sonication with an
400
-
300
o
u
200
100
12
Conc.of
11 10
mltogen,
9
8
7
-log(M)
Fig. 1. Dose-dependent stimulation of CE48T/VGH cell proliferation by EGF,
TGF-«. and IGF-1. Cell proliferation studies were performed as described in
"Materials and Methods." Cell numbers were determined in triplicate after a 4day treatment with different doses of EGF (•),TGF-a (O), or IGF-1 (*). Data
are expressed as the mean ±SD and are representative results from six independ
ent studies. SF, serum-free.
time of CE48T/VGH cells was estimated to be 140 h (data not shown).
The exogenous addition of these growth factors resulted in marked
proliferation of CE48T/VGH cells, in a dose-dependent manner. The
half-maximal stimulatory concentrations were 0.05 nM for EGF and
TGF-a and 0.1 nM for IGF-I. The maximal stimulation of cell prolif
eration by EGF or TGF-a treatment was 4.1-fold, compared with the
serum-free control, and that by IGF-I treatment was 3.6-fold. It has
centrifugation at 100,000 x g for 1 h. The solubilized membrane
been reported that both EGF and TGF-a bind to the EGF receptor and
fraction was purified with a wheat germ agglutinin affinity column and
stored at -70°C.
share many biological activities (for review, see Ref. 22). Our results
show that EGF and TGF-«are equipotent in the stimulation of CE48T/
Immunoprecipitation of Surface Receptors. For metabolic labeling,
VGH cell growth and that TGF-a competes equally with EGF for the
subconfluent monolayer cells were washed 3 times with HBSS and
labeled for 4 h in methionine-free DMEM containing 50 ^Ci/ini [>5S]- surface EGF receptor (data not shown).
CE48T/VGH Cells Overexpress EGF and IGF-I Receptors.
methionine and 10% dialyzed FCS. Crude membrane fractions were
prepared as described above and precleared with Protein A-Sepharose
The EGF and IGF-I receptors on CE48T/VGH cells were
coupled with control antibody, followed by incubation for 4 h at 4°C characterized by using 125I-ligand binding assay and Scatchard
with Protein A-Sepharose coupled with receptor-specific antibodies.
analysis. The number of EGF receptors was estimated to be 6.1
After extensive washing with NET buffer (150 mM NaCl, 0.5 mM x lO'/cell, with a Kj of 0.4 HM, and that of IGF-I receptors on
EDTA, 50 mM Tris, 0.25% Nonidet P-40, 0.1% BSA, 2 mM PMSF,
CE48T/VGH cells was estimated to be 2.5 x 105/cell, with a
pH 7.4), the immunocomplexes were boiled for 5 min in sample buffer,
Kj of 0.9 nM (Table 1). Both EGF and IGF-I receptors were
containing 0.08 MTris, 15% glycerol, 2% SDS, and 0. l Mdithiothreitol,
overexpressed in CE48T/VGH cells, compared to human lung
and electrophoresed on 10% SDS-PAGE.
In Vitro Autophosphorylation of the Receptors. Forty fig of solubi
lized membrane fractions of CE48T/VGH cells in kinase buffer (12
Table 1 EGF and IGF-I receptor numbers expressed on CE4STIVGH and other
mM MgSO4, 3 mM MnCh, 1 mM dithiothreitol) were treated with EGF
cell lines
(final concentration, 10~7M), IGF-I (10~7 M), or 10-fold concentrated
'"I-ligand binding assay was performed as described in "Materials and Meth
ods." The specific activities of '"I-EGF and '"I-IGF-I were 150-200 >iCi/^g. For
SFCM, at room temperature. After 15 min, 20 nCi of [r-'2P]ATP (250
Artek Ultrasonic 2000 (15 s at 30% power output, 3 times), and the
homogenates were centrifuged at 500 x g for 5 min. The supernatants
were further centrifuged at 100,000 x g for 1 h. The resulting pellets
were resuspended in extraction buffer (50 mM HEPES, 2% Triton X100, pH 7.4), stirred gently at 4°Cfor 2 h, and then subjected to another
Minol/VCi) were added. Phosphorylation proceeded for another 15 min
and was terminated by a 10-fold dilution with phosphatase inhibition
buffer (1 mM sodium vanadate, 20 mM sodium pyrophosphate, 20 mM
sodium fluoride, 100 MMzinc chloride, 1 mM ATP). The samples were
immunoprecipitated with either antiphosphotyrosine antibody or antireceptor antibodies. The immunocomplexes were subjected to 10%
SDS-PAGE and autoradiography.
CE48T/VGH. A431. CaLu-1. and HA22T/VGH cells, duplicate wells in 24-well
plates were incubated with 1 ng/ml labeled ligand, with or without various
concentrations of unlabeled ligands (0.5 to 100 ng/ml). Total binding was deter
mined after lysis of cells with 0.2% SDS. Nonspecific binding was determined by
addition of a 1000-fold excess of unlabeled ligand during incubation. For W'I-38
fibroblasts, 10' cells/well in six-well plates were subjected to binding studies. The
results are representative of five independent experiments.
IGF-I receptor
EGF receptor
lineCE48T/VGH
Cell
RESULTS
EGF, TGF-a, and IGF-I Are Potent Mitogens for CE48T/VGH Cells.
The mitogenic effects of EGF, TGF-«, and IGF-I on CE48T/VGH
cells were examined (Fig. 1). Under serum-free conditions, the doubling
x
2.5 x
A43I
1.6 x
CaLu-1
1.3 x
HA22T/VGH
WI-38Number/cell6.12.5 x
(nM)0.4
10'
10'
10'
10'
10"Kd
x 10*
1.9 x IO4
I.I
0.7
3.0 x IO4
1.1
0.9
3.0
x
IO4
2.9
0.72.2
4.0
x
IO4A'j(nM)0.9
1.3Number/cell2.5
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EGF AND IGF-I RECEPTORS
AND AUTOCRINE STIMULATION
1
(B)
(A)
—11.0kb
2
3
-210kD
-175
10.5kb —
-135
I-« 7.0kb
5.8kb —
-105
2.8kb —
Fig. 2. Northern analyses of EGF and IGF-1 receptor genes. Polyadenylated
mRNA was extracted from cells cultured in F2D1 medium containing 5% FCS.
Five ng of the mRNA samples were loaded per lane, and the Northern blots were
hybridized with 32P-labeled human EGF receptor cDNA probe (A) or human
IGF-I receptor cDNA probe (B), under stringent conditions. Expression of the
.¡anni gene was probed as an internal control (data not shown). A431 in A was
exposed for a shorter time, in order to visualize the 2.8-kilobase aberrant tran
script generated by chromosomal translocation (17).
carcinoma cell line CaLu-1, human hepatocellular carcinoma
cell line HA22T/VGH, or normal human fetal WI-38 fibroblasts. The number of EGF receptors on CE48T/VGH cells
was about 4 times greater than the number on CaLu-1 and
HA22T/VGH cells and 20 times greater than the number on
WI-38 cells. However, the number of EGF receptors on
CE48T/VGH cells was less than that on A431 cells, a human
epidermoid carcinoma cell line which was reported to have 30to 100-fold amplified EGF receptor genes (17). In addition,
CE48T/VGH cells were found to exhibit a 6- to 12-fold greater
number of IGF-I receptors than any other cell line examined.
In spite of the abundant surface receptors, the affinities (KJ) of
both EGF and IGF-I receptors for their specific ligands were
similar to those previously reported (23).
When polyadenylated RNA was prepared and subjected to
Northern blot analysis, it was clearly shown that CE48T/VGH
cells overexpressed EGF and IGF-I receptor genes at the
mRNA level, compared to HA22T/VGH and WI-38 cells (Fig.
2). Expression of the /3-actin gene was determined as an internal
control. The mRNA levels in all samples were relatively the
same (data not shown). A431 cells also showed overexpression
of EGF receptor genes, as reported previously. The 2.8-kilobase
transcript in A431 cells was an aberrant form generated by
chromosomal translocation (17). In CE48T/VGH cells, the
sizes of the EGF receptor gene transcripts were 10.5 and 5.8
kilobases, while those of the IGF-I receptor gene were 11.0 and
7.0 kilobases. In addition, the receptor genes of CE48T/VGH
cells were neither amplified nor grossly rearranged at the DNA
level, as shown by Southern blot analysis (data not shown).
EGF and IGF-I Activate Their Specific Membrane Receptors,
as Indicated by in Vitro Autophosphorylation. To confirm that
CE48T/VGH cells express EGF and IGF-I receptor proteins,
[35S]methionine-labeled membrane fractions were immunoprecipitated with receptor-specific antibodies. The anti-EGF recep
tor antibody identified a M, 175,000 protein (Fig. 3, lane 2),
corresponding to the reported EGF receptor (24). The IGF-I
receptor-specific antibody immunoprecipitated
three protein
bands, of A/r 105,000, 135,000, and 210,000 (Fig. 3, lane 3).
Fig. 3. Characterization of membrane receptors using antireceptor antibodies.
Immunoprecipitation of ("S]methionine-labeled membrane fractions of CE48T/
VGH cells was performed using NS-1 (lane I), anti-EGF receptor antibody (lane
2), or anti-IGF-I receptor antibody (aIR-3) (lane 3). The immunocomplexes were
resolved by 10% SDS-PAGE, followed by autoradiography.
The molecular sizes of the two smaller bands are identical to
those of the ß-and a-subunits of the IGF-I receptor reported
previously (25). The M, 210,000 protein was demonstrated to
be the precursor of the IGF-I receptor by a pulse-chase experi
ment (data not shown).
In order to demonstrate that EGF and IGF-I receptors on
CE48T/VGH cells have ligand-specific tyrosine kinase activity,
in vitro phosphorylation was performed. Fig. 4 shows the ligand-induced phosphorylation patterns detected by antireceptor antibodies (Fig. 4, lanes 1-4) and antiphosphotyrosine
antibody (Fig. 4, lanes 5-8). IGF-I enhanced phosphorylation
of a M, 105,000 protein which was detected by anti-IGF-I
receptor antibody (aIR-3) (Fig. 4, lanes 1 and 2) and antiphos
photyrosine antibody (Fig. 4, lanes 5 and 7). The size of this
protein is identical to that of the 0-subunit of the IGF-I receptor
LaneLigandAb1
2IaIR-33 4EcxEGFR5678CM
EocPhospho
I
- 1y ros i ne
105kD-*
Fig. 4. Ligand-induced receptor autophosphorylation. In vitro receptor phos
phorylation induced by IGF-I (/), EGF (E), or serum-free conditioned medium
(CM) was performed as described in "Materials and Methods." Equal amounts
of membrane fractions were incubated with fresh serum-free medium only (lanes
1, 3 and J) or activated by IGF-I (lanes 2 and 7), EGF (lanes 4 and *), or 10-fold
concentrated SFCM (lane 6). Phosphorylated receptors were immunoprecipitated
with aIR-3 (lanes 1 and 2), anti-EGF receptor antibody (lanes 3 and 4), or
antiphosphotyrosine antibody (lanes 5-8). The immunocomplexes were resolved
by 10% SDS-PAGE, followed by autoradiography.
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EGF AND 1GF-I RECEPTORS AND AUTOCRINE STIMULATION
Table 2 SFCM stimulation of CE48T/ VGH cell proliferation
Preparation of SFCM and assay for cell proliferation were carried out as
described in "Materials and Methods." Concentrated SFCM was diluted 1:40,
1:80, 1:160, and 1:320 with SF-F2D1 medium and added to CE48T/CGH cells
24 h after serum starvation. The SFCM was refreshed once after 2 days. Viable
cells were counted 4 days after the addition of SFCM. Values are expressed as
the mean ±SD. Percentage increase was determined as the increase in cell growth
in the SFCM-treated group, as compared with the serum-free medium group.
Cell number
(x IO4)
Increase (%)
Dilution of SFCM
Serum-free
medium
1:320
1:160
1:80
1:40
6.3 ±0.4
11.3 ±0.7
13.7 ±0.7
13.9 ±0.4
15.4 ±0.8
79
117
120
144
of
antibody(pg/ml)12.5
(25). EGF induced phosphorylation of a M, 175,000 protein
which was detected by anti-EGF receptor antibody (Fig. 4, lanes
3 and 4) and antiphosphotyrosine antibody (Fig. 4, lanes 5 and
8), corresponding to the EGF receptor identified in Fig. 3, lane
2. The phosphorylation sites were mainly on the tyrosine resi
dues, since the amount of phosphoprotein precipitated by re
ceptor-specific antibodies was not higher than that precipitated
by antiphosphotyrosine antibody.
Serum-free Conditioned Medium Promotes Cell Growth and
Enhances Autophosphorylation of EGF and IGF-I Receptors.
Since CE48T/VGH cells could propagate slowly in SF-F2D1
medium and the growth was dependent on cell density (data
not shown), CE48T/VGH cells may produce and respond to
endogenous growth factors in an autocrine manner. SFCM was
tested for the presence of mitogenic activity. When various
concentrations of SFCM were added to fresh SF-F2D1 me
dium, CE48T/VGH cell growth increased in a dose-dependent
manner (Table 2). Furthermore, SFCM enhanced autophosphorylation of two proteins of M, 175,000 and 105,000, cor
responding to the EGF receptor and the /3-subunit of the IGFI receptor (Fig. 4, lanes 5 and 6), suggesting the presence of
EGF-like and IGF-I-like activities in SFCM.
CE48T/VGH Cells Express TGF-a and IGF-I Genes. In order
to identify the growth factors which were produced by CE48T/
VGH cells, we examined the expression of growth factor genes,
by Northern blot analysis. Fig. 5 clearly demonstrates the
expression of TGF-a and IGF-I genes in CE48T/VGH cells,
with transcript sizes of 4.5 and 5.0 kilobases, respectively.
However, expression of the EGF gene was not detectable.
Anti-IGF-I Receptor Antibody Significantly Inhibits CE48T/
VGH Cell Growth. To further investigate whether the growth
factors synthesized by CE48T/VGH cells indeed stimulate their
(A)
Table 3 Inhibitory effect of anti-lCF-I receptor antibody on CE48T/VGH cell
growth
Cells seeded in 24-well plates were serum starved for 24 h, as described in
"Materials and Methods," followed by incubation with SF-F2D1 medium con
taining 12.5 to 100 Mg/ml anti-IGF-I receptor antibody (aIR-3). Monoclonal
antibody 9B2, which was also an IgG 1 antibody, against a bile canalicular antigen
(26), served as a control. Cell numbers were determined 4 days after the addition
of antibodies and are expressed as the mean ±SD. Percentage inhibition was
determined as the reduction in cell growth in the aIR-3 group, as compared with
the control antibody group.
IO4)Concentration
Cell number (x
(B)
-5.0kb
4.5kb-
TGFoc IGF-I
Fig. 5. Northern blot analyses of TGF-a and IGF-I gene expression Five /¿g
of
polyadenylated mRNA were subjected to Northern blotting and hybridized with
32P-labeled human TGF-«cDNA (A) and IGF-I cDNA (B) probes.
25
50toonIR-38.9
antibodv
(9B2)14.6
±0.5
7.9 ±0.1
16.4
6.4 ±0.3
20.3
6.9 ±0.4Control 21.2
±1.3
±0.5
52
±0.7
68
±1.2Inhibition(%)38
67
own growth, a proliferation-inhibition
assay was performed.
Anti-IGF-I receptor antibody (aIR-3), which is known to block
IGF-I binding to its receptor (25), significantly inhibited
CE48T/VGH cell growth in a dose-dependent manner under
serum-free conditions, compared to the effect of a control
antibody, 9B2 (26), against a bile canalicular antigen, which
was not expressed in CE48T/VGH cells (Table 3). The presence
of control IgG in the medium improved the culture conditions
for cell growth. This phenomenon was also observed when
another control antibody, L243 (anti-MHC class II), or BSA
was used (data not shown). At the concentration of 12.5 Mg/ml,
aIR-3 inhibited CE48T/VGH cell growth by 38%, and 50 ng/
ml aIR-3 inhibited cell growth by 68%. A further increase in
aIR-3 did not produce greater inhibition of cell growth. The
results strongly suggest IGF-I as an autocrine growth factor for
CE48T/VGH cells. Since an anti-EGF receptor antibody which
can efficiently block ligand binding is currently unavailable, a
similar proliferation-inhibition experiment using anti-EGF re
ceptor antibody was not performed.
DISCUSSION
Esophageal carcinoma exhibits striking geographic variations
in incidence. Various etiological agents have been suggested as
being the causative factors of this disease (27). However, the
molecular mechanism of oncogenesis and the biological prop
erties of esophageal carcinoma cells are poorly understood. Cell
lines have been established by us (12) and by others (28, 29)
from human esophageal tumor specimens to facilitate the ex
amination of the biological properties of this tumor in vitro,
thereby contributing to the better understanding
of esophageal
carcinogenesis as well as cellular regulation and differentiation.
In this study, we found that EGF, TGF-a, and IGF-I were
potent mitogens for a human esophageal carcinoma cell line,
CE48T/VGH.
These growth factors promoted cell proliferation
at physiological concentrations,
which may reflect their growthregulatory roles in vivo. We also demonstrated
that EGF and
TGF-a were equipotent in the stimulation of CE48T/VGH
cell
growth and that TGF-a competed equally with EGF for binding
to the surface EGF receptor. These results indicate that EGF
and TGF-« bind to the EGF receptor and may promote similar
biological activities in human esophageal carcinoma cells. Fol
lowing the interaction of EGF or IGF-I with the solubilized
cell membrane receptors, the EGF and the IGF-I receptors were
found to be autophosphorylated.
These results indicate that the
binding of EGF and IGF-I to CE48T/VGH
cells activates the
receptor-associated
protein tyrosine kinases and transduces the
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EOF AND IGF-1 RECEPTORS
AND AUTOCRINE STIMULATION
stimulatory signals of the growth factors, leading to activation
of gene expression and, ultimately, cell proliferation (30).
The results of Scatchard and Northern blot analyses indicated
that both EGF and IGF-I receptors were overexpressed in
CE48T/VGH cells, compared to human fibroblasts, lung car
cinoma cells, and hepatocellular carcinoma cells. At the DNA
level, neither apparent amplification nor truncation of these
receptor genes could be detected. Therefore, the overexpression
of EGF and IGF-I receptors is regulated at the mRNA level.
Overexpression of the EGF receptor has been suggested to be
related to carcinogenesis (1-6). Using epidermal cells that
express different amounts of surface EGF receptors, Boonstra
et al. (31) have shown that cells possessing high levels of surface
EGF receptors tend to have a decreased ability to enter the
terminal pathway of differentiation. Recent studies using
NIH3T3 cells have also indicated that overexpression of EGF
receptors can cause EGF- or TGF-a-induced malignant trans
formation (32, 33). Thus, the increase in surface EGF receptor
levels discovered in this study and others (5, 6) seems to be
associated with the development of human esophageal
carcinomas.
IGF-I stimulates growth of many types of cells, both in vitro
and in vivo (34, 35), through IGF-I receptors. The role of IGFI and its receptor in promoting tumor growth in vivo is unclear.
Recently, Kaleko et al. (36) demonstrated that overexpression
of human IGF-I receptors could trigger or potentiate IGF-Idependent neoplastic transformation of NIH3T3 cells. While
overexpression of IGF-I receptors on tumor cell lines has not
been documented, we found that CE48T/VGH cells expressed
a high level of IGF-I receptors, as demonstrated by Northern
blot analysis and ligand binding assay. Our study provides the
first example illustrating the role of IGF-I in carcinogenesis of
esophageal cells. Furthermore, the finding of overexpression of
both EGF receptors and IGF-I receptors on the same cells may
add more information on the mechanism of growth regulation
of malignant cells.
Alterations in the synthesis of endogenous growth factors
may result in uncontrolled growth and neoplastic transforma
tion. TGF-«and IGF-I have been identified as autocrine growth
factors in human carcinoma cells (6, 11). In this study, we found
that CE48T/VGH cells expressed TGF-«and IGF-I. In addi
tion, SFCM obtained from CE48T/VGH cells promoted cell
proliferation and induced autophosphorylation
of EGF and
IGF-I receptors. Our findings strongly suggest the existence of
TGF-« and IGF-I autocrine regulation in CE48T/VGH cells.
The observation that antibody to IGF-I receptor was able to
suppress cell growth under serum-free conditions further sub
stantiated the autocrine regulation by IGF-I of the growth of
CE48T/VGH cells. Therefore, through the overexpression of
EGF/TGF-« and IGF-I receptors on the cell surface and auto
crine synthesis of TGF-« and IGF-I in CE48T/VGH cells,
these growth factors may concertedly play an important role in
the development and progression of human esophageal
carcinoma.
Levine. G. Van de Woude, W. Topp, and J. D. Watson (eds). Cancer Cells,
pp. 5-10. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1984.
2. Y'amamoto, T.. Ramata, N., Kawano, H., Shimizu. S., Kuroki, T., Toyosh-
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
ACKNOWLEDGMENTS
We wish to thank Dr. Steve Roffler for reading the manuscript,
Shiang-lien Lu for preparation of the manuscript, and Dr. Kwen-jen
Chang for helping us to obtain «IR-3antibody.
22.
23.
24.
25.
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Overexpression of Epidermal Growth Factor and Insulin-like
Growth Factor-I Receptors and Autocrine Stimulation in Human
Esophageal Carcinoma Cells
Shan-Chun Chen, Chen-Kung Chou, Fen-Hwa Wong, et al.
Cancer Res 1991;51:1898-1903.
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