(CANCER RESEARCH 50. 1201-1205. February 15. 1990]
Modulation of Epidermal Growth Factor Receptors by Retinoic Acid
in ME180 Cells'
Zai-Sheng Zheng and Lowell A. Goldsmith2
Department of Dermatology; University of Rochester Medical Center, Rochester, New York 14642
stituents of the circulating blood and tissues, studies on the
effect of these two agents on various transforming cell lines
may provide an insight into the regulatory mechanism by which
proliferation, differentiation, and conversion to transformed
phenotypes occur. Retinoic acid specifically increases the num
ber of EGF receptors in various fibroblastic and epidermal cell
lines (11, 12). In this paper, we present evidence for the first
time that RA decreases the binding of 125I-EGF to its receptors
in ME 180 cells, a human epidermoid carcinoma, by reducing
the synthesis of EGF receptors.
ABSTRACT
Retinoic acid (RA) increases epidermal growth factor (M.I) receptors
in many cells; in ME180 cells, a human epidermoid carcinoma, RA
resulted in a dose- and time-dependent reduction of EGF binding. In RAtreated ME180 cells, binding was 41% of the control. The reduction of
EGF binding was due to a decrease in the number of receptors, from 8.7
x IO4 to 3.6 X IO4 per cell. The difference was present 8 h after the
addition of RA and was reversible 3 days after its removal. Scatchard
analysis indicated that RA did not change the binding affinity of EGF
(A',,= l UM). Also, RA did not alter the rate of EGF internalization or
the down-regulation induced by exogenous EGF. Flow-cytometric analy
sis revealed that RA did not alter the cell cycle. Soluble cell membrane
extracts were prepared in a Tris buffer with protease inhibitors, immunoprecipitated, electrophoresed, and immunoblotted with an antiserum
to EGF receptors. The EGF receptor band of M, 170,000 was decreased
in RA-treated cells. These results suggest that RA reduces the synthesis
of EGF receptors in M E180 cells.
MATERIALS
INTRODUCTION
Epidermal growth factor, a single-chain polypeptide (M, =
6045), is a potent mitogen in epidermal tissues in vivo and
induces proliferation in many cultured cells (1). It inhibits
proliferation of A431, a human epidermal carcinoma (2), GH4
rat pituitary tumor (3), and certain human breast cancer cells
(4). EGF-1 binds to plasma membrane receptors in a specific
and saturable manner. The binding of peptide hormone to
specific receptor sites on the plasma membrane is the first step
in initiating a biological response (5). After binding, EGF and
its receptors are clustered, internalized, and degraded. Further
more, in many tissue culture systems the introduction of EGF
produces a decrease in the number of EGF receptors. This
phenomenon is referred to as "down-regulation." The study of
EGF and its receptors has been intensified by the discovery that
EGF promoted tumor induction on mouse skin and enhanced
the tumorigenesis induced by a chemical carcinogen (6). In
primary squamous cell carcinomas, there is a frequent overexpression of EGF receptor kinase and erb-B mRNA relative
to normal tissues, and in some instances this is due to amplifi
cation of the EGF receptor gene (7), which is a protooncogene
coding for the EGF receptor. Elevated EGF receptor expression
was found in 100% of the lung, vulval, and cervical carcinomas
examined (8, 9).
On the other hand, RA has been shown to have an anticarcinogenic effect. Retinoids inhibit the increased cell prolifera
tion and metaplasia typically triggered by exposure to chemical
carcinogens (10). Since EGF and vitamin A are normal conReceived3/29/89; revised9/28/89; accepted 11/9/89.
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.
1Supported by USPHS Grants AR30126 and AR30965. Presented in part at
the Society for Investigative Dermatology annual meeting. May, 1988 (J. Invest.
Dermatol., 90:620, 1988).
2To whom requests for reprints should be addressed, at University of Rochester
Medical Center, 601 Elmwood Avenue. P.O. Box 697. Rochester. NY 14642.
3The abbreviations used are: EGF, epidermal growth factor; BSA, bovine
serum albumin; PCS, fetal calf serum; PBS. phosphate-buffered saline; RA,
retinoic acid; TGF, transforming growth factor; RPMI, RPMI-1640 medium;
SDS, sodium dodecyl sulfate; PAG, polyacrylamide gel.
AND METHODS
Materials. Tissue culture media, Dulbecco's modified Eagle's me
dium and RPMI-1640, were purchased from Hazelton (Lenexa, KS).
Fetal calf serum was obtained from Hyclone (Logan, UT). Tissue
culture plasticware was from Corning (Corning, NY) or Costar (Cam
bridge, MA). Receptor grade EGF was purchased from Sigma (St.
Louis, MO). L-[15S]Methionine and carrier-free Na'"I for protein iodination were obtained from Amersham (Arlington Heights, IL). lodination of EGF was by a chloramine-T procedure (13) to a specific
activity of 1400-1700 Ci/mmol, using a low concentration of chlora
mine-T (2 Mg/60-M' reaction), which does not activate the iodinated
EGF to a product that cross-links to receptors. Antiserum for the EGF
receptor (no. 451) was a generous gift from Dr. Christa M. Stoscheck,
Vanderbilt University School of Medicine. All other chemicals were of
the highest grade available.
Cell Culture. Human epidermoid carcinoma cell line ME 180 was
isolated from an omental metastasis of a spreading cervical carcinoma
by J. A. Sykes and associates in 1967. It was a generous gift from him
and has been sustained in our laboratory since 1978. A431 and 3T3,
both obtained from American Type Culture Collection (Rockville, MD)
and maintained in Dulbecco's modified Eagle's medium supplemented
with 10% PCS, and ME180 cells, maintained in RPMI-1640 supple
mented with 10% FCS, were grown in a humidified atmosphere of 95%
air/5% CO2 at 37°C.Cultures were Mycoplasma-free, testing negative
with bisbenzimide H 33258 fluorochrome from Calbiochem (San
Diego, CA).
EGF Binding. To measure binding, '"l-EGF was added to 1 ml of
RPMI-1640 without FCS. After incubation for the times described in
the text, the medium was aspirated, and the dishes were washed three
times with cold PBS + 0.1% BSA at 4°C.Cells were scraped into 1 ml
of 0.1 N NaOH. Samples of the solubilized cells were counted in a
Beckman 5500 gamma counter. Nonspecific binding was determined
in parallel cultures which had a 200-fold excess of unlabeled EGF.
Nonspecific binding was 4-10% of the total binding and has been
subtracted from all points. The average difference between determina
tions on duplicate or triplicate dishes was less than 8%. Cells were
counted with a Coulter Counter. To distinguish surface-bound from
internalized EGF, we used a technique that has been reported to
measure the distribution of 1!5I-EGF between cell surface receptors and
intracellular sites (14). In this technique, cells that had been incubated
with '"I-EGF were first washed to remove radioactivity not bound to
the receptor and then incubated for 5 min at 0°Cwith 0.2 M acetic acid0.5 M NaCl (acid-salt wash) to remove '"I-EGF bound to the cell
surface receptors. The '"I-EGF that is not removed with this buffer is
considered to be ligand which has been internalized. The acid-salt
solution was measured to obtain the amount of radioactivity bound to
cell surface receptors.
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RETINOIC ACID AND EOF RECEPTORS
Metabolic Labeling of the EGF Receptor and Solubilization of Intact
Cells, ubconfluent ME180 cells (85% of the dish area) were grown in
35-mm dishes containing RPMI-1640 supplemented with 10% FCS.
For radioisotope incorporation, the cells were washed twice with PBS
and then incubated for 24 h in methionine-free growth medium con
taining 3% FCS with 25 ^Ci/ml L-["S]methionine (1420 Ci/mmol)
and I0~5 M RA in 0.1% ethanol or 0.1% ethanol alone. Cell protein
was quantitated in replicate dishes not containing radioisotope. To
prepare solubilized cell extracts for immunoprecipitation and inimiinoblotting, the cells were washed three times with PBS and solubilized
for 20 min at room temperature in 1 ml RIPA buffer (10 m\i Tris. pH
8.5, 0.15 M NaCl, I mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 200 KIU/ml aprotinin, and 0.1 mg/ml phenylmethylsulfonyl fluoride)/mg of cell protein. Insoluble material was
removed by centrifugation at 140.000 x g for 40 min at 4°C,and the
supernatants were subjected to immunoprecipitation and immunoblotting. The labeled receptor was immunoprecipitated with rabbit antihuman serum according to the method of Stotcheck and Carpenter
(15). The EGF receptor was precipitated from 0.5 mg of cell extract
using 2 ¿il
antiserum and 40 n\ of a 50% protein A-Sepharose suspension
from Pharmacia.
Electrophoresis and Autoradiography. Immunoprecipitated samples
were boiled for 5 min in sample buffer and electrophoresed on a 7.5%
SDS-PAG. Molecular weight markers were purchased from Bio-Rad
(Rockville Centre, NY), and the gel was stained with 0.05% Coomassie
G-250. For autoradiography, the gels were first dried on a Bio-Rad
model 224 slab gel drier and then exposed to film at —¿70°C.
of EGF binding compared to control after 2 h. The diluent,
0.1% ethanol. did not change the EGF binding.
The relationship between EGF binding and the concentration
of RA administered was determined in ME 180 cells pretreated
for 24 h with 0-10-5 M RA (Fig. 2). The level of EGF binding
was dependent on the concentration of RA, with a significant
(P < 0.01) decrease detected at concentrations greater than
10~" M. The data in this study were obtained in the absence of
serum, indicating that RA acts directly on ME 180 cells rather
than affecting or being affected by serum components.
The magnitude of EGF binding as a function of time with
RA was determined. Fig. 3 shows the saturable EGF binding
in cells exposed to 10~5 M RA, and the control, for 0-24 h. A
significant (P < 0.05) decrease in EGF binding was detected in
treated cells after 8 h. This effect was reversible. The decreased
binding detected in cells treated with 10~5M RA for 24 h began
to return toward normal after 24 h, reaching control levels after
3 days of growth in RA-free medium (Fig. 4).
To examine the role of RA in the regulation of EGF receptor
levels, monolayer ME 180 cells were incubated in serum-free
RPMI with and without IO'5 M RA for 24 h. The results of
saturation analysis for EGF binding to ME 180 cells incubated
in IO"5 M RA-treated and control media are illustrated in Fig.
5. Scatchard analysis indicated that RA did not change the
Immunoblotting. Replicate samples not containing radioisotope (60
ng protein/well) were electrophoresed on a 7.5% SDS-PAGE. After
transferring, one sheet of nitrocellulose was immunoblotted with EGF
receptor antiserum (1:1000) for 30 min, incubated with biotinylated
secondary antibody for 30 min, and then reacted with ABC reagent
from Vector Laboratories (Burlingame, CA). The other was stained
with 0.2% Amido black for protein.
40â„¢¿
o
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£
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3530 25 20-
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D-
RESULTS
Z
The time required for maximal binding of EGF was investi
gated to ensure that these studies were performed under equi
librium conditions. ME 180 cells were treated for 24 h with 10 5
M RA or its diluent, 0.1% ethanol (control), and saturable
binding was measured after 0.5-6 h at 4°C.Fig. 1 shows that
EGF binding plateaus after 2-6 h in both groups of cells,
suggesting that equilibrium is reached by this time. A 3-h
incubation time was used in all subsequent experiments. These
data also show that EGF binding was decreased in ME 180 cells
treated with 10~5 M RA at all times measured, with only 30%
10
OM
10 9 M
10 8 M
IO'7 M
IO6
M
10 5 M
RA Concentration
Fig. 2. Effect of RA concentrations on EGF binding. Monolayer confluent
ME180 cells were treated with 0-10~' M RA (*) or without RA (O) for 24 h. The
EGF binding was assayed at 3.8 x 10*cpm/ml at 4'C for 3 h, as described under
"Materials and Methods." Bars. SD.
o
10
12
14
16
18
20
22
24
Time (hours)
Time (hours)
Fig. 1. Timecoursc for EGF binding. M E180 cells (4 x lO'/dish) were seeded
in 35-mm dishes containing 2 ml RPMI with 1(K¿FCS, grown for 24 h. rinsed
with 2 ml PBS. and then incubated for 24 h in RPMI without FCS in the presence
of I0~* M RA in 0.1% ethanol (*) orO. 1% ethanol as a control (O). After removal
of media, cells were rinsed twice using PBS + 0.1% BSA. and then '"I-EGF. }
X 10' cpm/dish. was added. The EGF binding was assayed as described under
"Materials and Methods."
Fig. 3. Time course of '"I-EGF binding to ME 180 cells in the presence and
absence of I0~' M RA. Monolayers of ME180 cells in 35-mm dishes were
incubated in RPMI with I0~' M RA (»)or without RA (O) at 37'C. After the
indicated times, cells were rinsed three times with PBS+ 0.1% BSA. Fresh RPMI
(1 ml) containing 3 x 10'cpm of'"I-EGF was added to every dish and incubated
at 4°Cfor 3 h. All dishes in duplicate were washed three times with 2 ml cold
PBS + 0.1% BSA. solubilized with 0.1 M NaOH. and counted in a gamma
counter.
1202
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RETINOIC ACID AND EOF RECEPTORS
n i
3500
Q
Control
D
RA (10-5 M)
Acid Resistant
3000
2500
2000
1500
1000
500
Acid Releasable
0
5
10
15
20
25
30
Time (minutes)
Days After RA Removal
Fig. 4. Reversal of RA effect. Approximately 5 x 10s cells/35-mm dish were
seeded. At 24 h after seeding, cells were divided into two groups. The first group
was incubated in RPMI with I0~' M RA and the second group with 0.1% ethanol
for 24 h. After removal, the cells were washed twice with
ml RPMI + 5% PCS were added. The binding of '"I-EGF
cells of both groups was determined on the indicated
"Materials and Methods." Cell counts were performed on
2 ml PBS. and then 2
(3 x 10'cpm/dish) to
days as described in
parallel cultures.
B
Binding (Imol well)
Fig. 5. Binding of'"I-EGF to cultured ME 180 cells. ME 180 cells were grown
in 80-cmJ flasks and subcultured at high density (1.5 x 10' cells/well) in 24-well
trays. After 24 h of incubation in RPMI with 10~' M RA (»)or its diluent, 0.1%
ethanol. as control (O). monolayers of confluent ME180 cells were rinsed two
limes with PBS + 0.1% BSA. Cells were then incubated for 3 h at 4'C in the
presence of increasing concentrations of '"I-EGF in a final volume of 0.25 ml
RPMI. Data were then analyzed by the method of Scatchard using three wells/
point. Nonspecific binding used 200-fold excess unlabeled EOF. Bound/free ratios
are indicated, and nonspecific binding was 3.2-7%. Wells contained approxi
mately 3 x 10' cells in 10~' M RA-treated cultures and 3.1 x 10' cells in the
Time (minutes)
Fig. 6. A. distribution of cell-associated '"I-EGF as a function of time.
Monolayers of MEI80 cells were incubated 2 days until 85% confluent. Fresh
RPMI with 10~' M RA (»)or its diluent. 0.1% ethanol (O), was then added.
Incubation was continued for 24 h until 100% confluent. Monolayers were then
incubated with '"I-EGF (4.9 x 10' cpm/dish) for 3 h at 0°C.The dishes »ere
aspirated and rinsed three times with ice-cold PBS + 0.1% BSA to remove
unbound '"I-EGF. Fresh RPMI (1 ml) preequilibrated at 37°Cwas added to the
dish. Following incubation at 37°Cfor the indicated times, medium was removed
and counted in a gamma counter. The '"I-EGF binding only to surface receptors
of ME180 cells was measured by acid-salt washes: then 1 ml 0.1 M NaOH was
added. Specifically bound '"1-EGF, resistant to the acid-salt solution, was
counted. B. results expressed as the relative percentage of bound radioactivity,
taking 100% as the total cell-associated radioactivity present after the initial 3 h
at O'C.
controls.
binding affinity of EGF (Kd = 1 nM). The reduction of EGF
binding was due to a 59% decrease in the number of receptors,
from 8.7 x IO4 to 3.6 x 10" per cell.
We used a technique that has been reported to measure the
distribution of I25I-EGF between cell surface receptors and
intracellular sites (14). ME 180 cells were incubated in media
with 10~5M RA or 0.1% ethanol for 24 h. The rate of internalization of EGF was measured by incubating these two groups
with '"I-EGF for 3 h at 0°C,to allow binding to proceed to
equilibrium without internalization. The cells were then rinsed
to remove unbound hormone and incubated at 37°Cfor various
times before measuring cell-associated radioactivity with the
acid-salt wash. Only 15% of the cell-associated '25I-EGF was
still released by washing after 20 min at 37°C,indicating that
internalization of '"I-EGF proceeds rapidly at 37°Cin both
Fig. 7. Time course of EGF receptor down-regulation. The IO~!M RA-treated
(O) and control (O) ME180 cells were incubated at 37'C in RPMI containing 2
nvi EGF. At different time intervals, cells were washed three limes with RPMI,
and the '"I-EGF binding (3 x 10' cpm/ml) was determined at 4'C for 3 h.
samples. There is no difference in the rate of internalization
between 10~5 M RA-treated and control ME 180 cells (Fig. 6).
treated to control ME 180 cells by measuring the binding of
I25I-EGF to its available receptor sites at various times after
Ti me (hours)
exposure to 2 nM EGF. The experiment was designed so that
To determine whether RA induced an increased transient
down-regulation of EGF receptors similar to the effect of EGF
the incubations with EGF terminated at the same time. As
receptors on 3T3 cells by platelet-derived growth factor (16), shown in Fig. 7, a dramatic reduction in I25I-EGF binding
we compared the down-regulation of EGF receptors in RA- capacity occurred after prior incubation with 2 nM EGF. Re1203
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RETINOIC ACID AND EGF RECEPTORS
ceptor loss in 10 5 M RA-treated cells was 89% of the total
binding and 90% in the control cells, suggesting that there is
no significant difference in EGF-induced down-regulation.
Incubation for 24 h with 10~5 M RA has little effect on cell
growth rate (3-5% decrease). Therefore, we took 24-h incuba
tions in these experiments to avoid the reduction of cell number
resulting from long exposure to RA. Flow-cytometric analysis
revealed that the cells in S phase accounted for 22.2% of total
in RA-treated ME 180 cells, compared with 20.9% in the con
trol. In both the control and treated cells, G2/G, = 1.91 (data
not shown). It is clear that in this study RA did not affect the
cell cycle, which is a potential cause of receptor reduction.
It is reasonable to postulate that RA might cause ME 180
cells to produce some unknown factor, such as TGF-«, which
competes for available EGF binding sites and results in a
decrease in EGF binding. Therefore, ME 180 cells were incu
bated for 24 h in 10~5 M RA or ethanol. We used this condi
tioned medium to test whether there were some factors induced
by RA. The binding of EGF was determined in the presence of
1 ml of these conditioned media or an RPMI control each with
50 M'of 125I-EGF (3 x 10' cpm/dish). Conditioned media with
RA did not affect cells during this 3-h incubation. The binding
sites were 5758 ±576 cpm/106 cells in RPMI, 5625 ±465
cpm/IO6 cells in ethanol, and 5809 ±179 cpm/106 cells in RAconditioned medium. There was no significant difference.
We studied the action of RA on two other cell lines (Table
1). It is interesting that the same concentration of RA, which
decreased the EGF binding to ME 180 cells, stimulated 2-fold
the binding of EGF to 3T3 cells and had little effect on the
binding to A431 cells.
Studies by others have shown that the EGF receptor protein
has a molecular weight of 150,000-170,000 (15). Fig. 8 shows
the presence of a major protein band at M, 170,000 in both
samples. A few trace proteins of lower molecular weight might
represent degraded or truncated EGF receptors. Densitometric
scan of this band, shown in RA-treated cells, is approximately
38% of ethanol control, based upon comparison of the intensity.
The same results were obtained from immunoprecipitation. Fig.
9 shows the results obtained with normal rabbit serum (A). No
EGF receptor band was found except nonspecific traces, prob
ably precipitated by Protein A-Sepharose. The EGF receptor
band at M, \ 70,000 precipitated in the control (B) is also shown
to be more intense than that in RA-treated cell extracts (C).
DISCUSSION
We have demonstrated here that RA results in a dose- and
time-dependent reduction of EGF binding at concentrations
greater than 10~8M in ME 180 cells. The effects on EGF binding
were present 8 h after exposure to RA and were reversible,
lasting as long as 3 days after the removal of RA. This was
Table I Effect ofRA on '"I-EGFbinding to 3T3. A43I, and MEI80 cells
The binding of '"I-EGF to different cell lines was determined as described in
"Materials and Methods." Each value is the mean ±SD of three determinations.
(cpm/106Cell
EGF binding
xControl5.8
43K
Fig. 8. Immunoblotting of the EGF receptor M, 170.000 band. Extracted cell
protein (60 /¿g/well)was electrophoresed on a 7.5cr SDS-PAG. transferred, and
immunoblotted as described under "Materials and Methods." Lanes C and D,
equal amounts of cell protein: Lane E, standard protein markers as stained by
Amido black. Lanes A and /*'.an immunoblotted A/r 170,000 band indicating the
EGF receptor. Lanes fi and D, from control ME 180 cell extracts; Lanes A and C.
from IO"5 M RA-trcated cell extracts. Intensity of the M, 170.000 bands in both
Lanes B and D is more than that seen in Lanes A and C.
170K
Fig. 9. Effect of RA on EGF receptor synthesis. ME 180 cells, prelabeled with
L-[35S]methionine. were scraped in RIPA buffer. Solubilization, immunoprecipi
tation, electrophoresis, and autoradiography were performed as described under
"Materials and Methods." The immunoprecipitatcs are shown in Lanes A, B and
C. Lane A, obtained with normal rabbit serum. Lane B. an A/r 170.000 band of
the EGF receptor immunoprecipitated by 2 nl of EGF receptor anliscrum in 0.5
mg of control extract. Lane C. a weaker M, 170.000 band immunoprecipitated
by 2 j.I of antiserum in 0.5 mg of RA-treated cell extract. Lane D. derived from
direct Solubilization of whole cells in RIPA buffer.
kinetically similar to findings in the study by Jetten (11),
although in his studies RA increased EGF receptors, whereas
in our studies receptors were decreased. The greatest effect of
reduction was observed at IO"5 M RA. Since in serum-free
cultures the detachment of ME180 cells from dishes increased
at concentrations greater than 10~5 M for more than 36 h,
experiments were performed at 10~5 M for a period of 24 h. In
treated/control1.9°0.960.36°
contrast,
RA did increase EGF binding by 2-fold in 3T3 cells
with the same conditions, which is consistent with Jetten's data
lineMouse
type and cell
fibroblasts3T3Human
0.435.8
±
200K
±0.834.4
epidermoidcarcinomaA431ME180cells)
1.34.7
±
0.0513.1
±
±0.3io-JRA(IO-'M)11.1
±0.2Ratio
(11). As mentioned above, our binding experiments were per
formed in a serum-free system, but the data of Jetten were
obtained in media with 10% FCS. Interestingly, with or without
FCS, RA increased the binding of EGF to 3T3 cells. Although
we did not study in detail the difference of EGF binding in the
presence and absence of FCS, it seems clear that RA acts
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RETINOIC ACID AND EOF RECEPTORS
directly on ME 180 cells rather than affecting or being affected
REFERENCES
by serum components.
1. Carpenter. G. The biochemistry and physiology of the receptor kinase for
It should be pointed out that RA does not bind to the EGF
epidermal growth factor. Mol. Cell. Endocrino!., il: 1-19, 1983.
receptor, nor does it share any structural similarity with EGF
2. Gill, G. N., and Lazar, C. S. Increased phosphotyrosine content and inhibi
tion of proliferation in EGF-treated A431 cells. Nature (Lond.), 293: Ì05(16). Studies indicate that growth factors receptors are not only
307, 1981.
modulated by their respective ligands but also by other growth
3. Schonbrunn, A.. Krasnoff. M., Westendorf. J. M.. and Tashjian, A. H.. Jr.
factors. For example, both platelet-derived growth factor and
Epidermal growth factor thyrotropin releasing hormone acts similarly on a
TGF-« regulate EGF receptors (17-19), and human a-interclonal pituitary cell strain. J. Cell Biol., S5: 786-797. 1980.
4. Imai, Y.. Leung. C. K. H., Friesen, H. G., and Shiu, R. P. C. Epidermal
feron appears to influence both receptor number and the bind
growth factor and effect of EGF on growth of human breast cancer cells in
ing affinity for EGF (20).
long-term tissue culture. Cancer Res., 42: 4394-4398. 1982.
A variety of human tumors secrete the related hormone TGF5. Bradshaw. R. A., and Frazier, W. A. Hormone receptors as regulators of
hormone action. Curr. Top. Cell. Regul.. 12: 1-37, 1977.
a, which acts through the EGF receptor and shows a 40%
6. Rose. S. P.. Stahn, R., Passovoy, S. D., and Herschman. H. Epidermal
structural homology with EGF (21). In our experiments, RAgrowth factor enhancement of skin tumor induction in mice. Experientia
induced growth factors, which bind to the EGF receptor, were
(Basel), ¿2:913-915, 1976.
7. Hunts, J., Ueda, M., Ozawa, S.. Abe, O., Pastan, I., and Shimizu, N.
not detected. To our knowledge there are no reports of retinoids
Hyperproduction and gene amplification of the epidermoid growth factor in
increasing TGF-a production, although the possibility of se
squamous cell carcinomas. Jpn. J. Cancer Res.. 76: 663-666, 1985.
creted growth factor, like TGF-«, being related to the mecha
8. Ozanne, B., Richards, C. S., Hendler, F., Burns, D., and Gusterson, B.
Growth factors in relation to epithelial neoplasia. J. Pathol., 146: A289.
nism of these changes cannot be ruled out completely. This
1985.
point can best be checked by detecting the mRNA of TGF-«
9. Gullick, W. J., Marsden, J. J., Whittle, N., Ward, B., Bobrow, L., and
(detailed studies in progress). Also, RA did not increase the
Waterfield, M. D. Expression of EGF receptors on human cervical ovarian
rate of internalization or down-regulation of the EGF receptor
and vulval carcinomas. Cancer Res., 46: 285-292, 1986.
10. Crocker, T. T., and Sanders, L. L. Influence of vitamin A and 3,7-dimethylcomplexes. Immunoprecipitation
experiments confirmed the
2,6-octadienal (citral) on the effect of benzo(a)pyrene on hamster trachea in
conclusion drawn from the Scatchard analysis, that RA de
organ culture. Cancer Res., 30: 1312-1318, 1970.
creased the number of total EGF receptors present in ME180
11. Jetten, A. M. Retinoids specifically enhance the number of epidermal growth
factor receptors. Nature (Lond.), 284: 626-629, 1980.
cells. Further studies have revealed that RA suppressed EGF
12. Komura, H., Wakimoto, H., Chu-Fung, C., Terakawa, N., Aono, T.. Tanireceptor gene transcription and mRNA levels (22), but other
zawa, O., and Matsumoto, K. Retinole acid enhances cell responses to
experiments including EGF receptor pulse labeling are neces
epidermal growth factor in mouse mammary gland in culture. Endocrinology.
118: 1530-1536, 1986.
sary to determine whether retinoids affect EGF synthesis in
13. Hunter, W. M., and Greenwood, F. C. Preparation of iodine-131 labelled
addition to altering levels of its message.
human growth hormone of high specific activity. Nature (Lond.), 194: 495For certain cell lines, EGF binding is strongly dependent on
496, 1962.
14. Haigler, H. T.. Maxfield, F. R., Willingham. M. C.. and Pastan, I. Dansylcell cycle (23) and cell density (24). Even though our binding
cadaverine inhibits internalization of '"I-EGF in BALB 3T3 cells. J. Biol.
experiments were performed on monolayer confluent dishes,
Chem., 255: 1239-1241, 1980.
the effect of cell cycle and density on EGF binding remains to 15. Stoscheck, C. M.. and Carpenter, G. Characteristics of antibodies to the EGF
receptor-kinase. Arch. Biochem. Biophys., 227: 457-468, 1983.
be studied more extensively, as does the exact mechanism by
16. Cope, F. O., and Boutwell, R. K. A role for retinoids and a tumor promoting
which RA modulates EGF receptors, the structural specificity
phorbol ester in the control of protein phosphorylation. In: Retinoid Sym
of retinoids for reducing EGF binding, and the effect of RA on
posium, Geneva, 1984, pp. 106-124.
other cell components besides the EGF receptor.
17. Wrann, M., Fox, C. F., and Ross, R. Modulation of EGF receptor on 3T3
cells by platelet-derived growth factor. Science (Wash. DC), 210: 1363-1365,
The regulation of EGF receptor levels is biologically impor
1980.
tant, since high expression of these receptors is a common
18. Collins, M. K. L., Sinnett-Smith, J. W., and Rozengurt, E. Platelet-derived
property of squamous cell carcinomas and sometimes occurs
growth factor treatment decreases the affinity of the EGF receptor of Swiss
3T3 cells. J. Biol. Chem., 25«:11689-11693. 1983.
during the progression of normal epidermal cells to a malignant
state. Although EGF has tumor-promoting activity in some cell 19. Coffey, R. J., Derynck, R., Wilcox, J. N., Bringman, T. G.. Goustin. A. S.,
Moses, H. L., and Pittelkow, M. R. Production and auto-induction of
systems (25) and induces cytogenetic damage in mammalian
transforming growth factor in human keratinocytes. Nature (Lond.), 328:
817-820, 1987.
cells (26), the receptor itself is related both structurally and
20. Zoon, K. C.. Karasaki, Y., Nedden, D. L.. Hu, R., and Arnheiter, H.
functionally to certain transforming proteins. A recent report
Modulation of EGF receptors by human a interferon. Proc. Nati. Acad. Sci.
has indicated a correlation between the invasiveness of human
USA, 83: 8226-8260, 1986.
bladder tumors and a high level of EGF receptor expression
21. Marquardt, H., Hunkapiller, M. W., Hood, L. E., and Todaro, G. J. Rat
transforming growth factor type I: structure and relation to epidermal growth
(27), suggesting that the receptor analysis may be useful in
factor. Science (Wash. DC). 22.?: 1079-1082, 1984.
predicting the behavior of a tumor. The complexity of hormonal
22. Zheng. Z. S.. Polakowska, R.. and Goldsmith, L. A. Retinoid suppression of
interactions, including EGF with cells, makes the elucidation
EGF receptor gene transcription in ME180 cells. J. Invest. Dermatol., 92:
548, 1989.
of their precise role in carcinogenesis difficult. The A431 cell
23. Fanger, B. O., Currie, A., and Cidlowski, J. A. Regulation of EGF receptors
line synthesizes a truncated form of the EGF receptor, com
by glucocorticoids during the cell cycle in HeLa S, cells. Arch. Biochem.
prised of the external glycosylated binding domain which is
Biophys.. 294: 116-125, 1986.
secreted into the growth medium (28). However, it should be 24. Brown, K. D., Yeh, Y. C., and Holley, R. W. Binding, internalization. and
degradation of epidermal growth factor by BALB 3T3 and BP 3T3 cells:
emphasized that there is no direct evidence that overexpression
relationship to cell density and the stimulation of cell proliferation. J. Cell.
or aberrant expression of EGF receptors is involved in the
Physiol., 100: 227-238, 1979.
transformation of human cells (9). Although little is understood
25. Kingsnorth. A. N.. Abu-Khalaf. M.. Ross. J. S., and Malt. R. A. Potentiation
of 1,2-dimethv 1hj drazine-induced anal carcinoma by epidermal growth factor
about the role in anticarcinogenic events, the action of retinoids
in mice. Surgery (St. Louis). 97: 696-700, 1985.
on EGF binding may provide a screening assay for potential
26. Kelsey, K. T., Nagasawa, H., Umans, R., and Little, J. B. Epidermal growth
activity in inhibiting carcinogenesis.
factor induces cytogenetic damage in mammalian cells. Carcinogenesis
ACKNOWLEDGMENTS
We would like to thank Dr. Patricia M. Hinkle for her scientific
input, Nancy Ewing for flow-cytometric analysis, and Vincent Falciano
for his assistance with this manuscript.
(Lond.), 8: 625-627. 1987.
27. Neal. D. E., Marsh, C, Bennett, M. K., Abel, P. O., Hall, R. R.. Sainsbury.
J. R. C., and Harris, A. L. Epidermal growth factor receptors in human
bladder cancer: comparison of invasive and superficial tumours. Lancet, 1:
366-368. 1985.
28. Weber, W., Gill, G. N., and Spiess. J. Production of an epidermal growth
factor-related protein. Science (Wash. DC). 224: 294-297. 1984.
1205
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
Modulation of Epidermal Growth Factor Receptors by Retinoic
Acid in ME180 Cells
Zai-Sheng Zheng and Lowell A. Goldsmith
Cancer Res 1990;50:1201-1205.
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