Download Epidermal Growth Factor Stimulation of Human

[CANCER RESEARCH 40. 2361-2366,
0008-5472/80/0040-OOOOS02.00
July 1980]
Epidermal Growth Factor Stimulation of Human Breast Cancer Cells in
Culture1
C. Kent Osborne,2 Barbara Hamilton, Grace Titus, and Robert B. Livingston
Department of Medicine,
Texas 78284
University
of Texas Health Science
Center ¡C. K O.. B. H.¡.and the Audie Murphy
ABSTRACT
Epidermal growth factor (EGF), a polypeptide found in human
and animal blood and secretions, has been found to stimulate
a variety of tissues in vitro including normal and malignant
rodent mammary epithelium and human breast epithelial cells
and fibroadenoma. We have studied the influence of EGF on
malignant human breast tissue with a model system comprising
human breast carcinoma cells growing in tissue culture. EGF
stimulated growth of MCF-7 cells in serum-free medium. After
7 days in culture, a 2-fold increase in cell number and DMA
content and a 3-fold increase in total protein were observed in
cells incubated with EGF (10 ng/ml). As little as 0.01 ng/ml of
EGF stimulated growth; 10 ng/ml was maximal. EGF effects
on growth were noted for cells plated at a high as well as
sparse (cloning) density. EGF also stimulated the rates of
thymidine, uridine, and leucine incorporation into macromolecules in a dose- and time-dependent fashion. Stimulation of
uridine and leucine incorporation was evident by 3 hr, whereas
EGF stimulation of thymidine incorporation was delayed until
12 to 18 hr. EGF increased the proportion of cells active in
DMA synthesis by nearly 2-fold. The combination of optimal
concentrations of insulin (also a growth factor for these cells)
and EGF did not stimulate growth above that seen with either
hormone alone, suggesting a common step in their mechanism
of action. The EGF effect was not dependent on the presence
of serum and was not enhanced by dexamethasone as reported
for other types of cells. EGF had no effect on another human
breast cancer cell line, the MDA-231. These studies suggest
that growth of some human breast cancers may be influenced
by EGF.
INTRODUCTION
EGF3 is a single-chain polypeptide
isolated first from mouse
submaxillary glands (7) and later from human urine (8). Al
though mouse EGF and human EGF differ slightly with regard
to molecular weight, amino acid composition, and immunological properties, they both compete for the same cell membrane
receptor and demonstrate similar biological activities (6). The
tissue site of EGF production and the regulation of its secretion
are not understood. However, it is known that daily urinary
excretion of EGF in humans exceeds 50 fig/day, is significantly
' This investigation was supported in part by Grant BC-288 from the American
Cancer Society. Grant CA 24129 awarded by the National Cancer Institute.
Department of Health, Education, and Welfare, and by a grant from the Ruth
Estrin Goldberg Memorial for Cancer Research.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: EGF, epidermal growth factor; TCA, trichloroacetic acid; IMEM, Richter's improved Eagle's minimal essential medium ZO; TLI,
thymidine labeling index.
Received September 17, 1979; accepted April 14. 1980.
JULY
VA Hospital ¡G. T.. R. B. L ]. San Antonio.
higher in females, and is 50% higher in women who are taking
oral contraceptives (9). In addition, EGF has been detected in
human plasma, human milk, and other secretions in measurable
concentrations (ng/ml) (9, 22). EGF appears to be closely
related if not identical to the gastric antisecretory hormone
urogastrone (12).
Although the relevance of EGF to normal human physiology
and disease has not been clarified, data suggest that this
polypeptide may be an important regulator of cell growth. EGF
is mitogenic for a panoply of ectoderm-, mesoderm-, and
endoderm-derived cells in tissue culture (11 ). However, not all
cells in culture respond to EGF, which suggests some target
tissue specificity and supports the notion that it may have a
physiological role in vivo.
Several studies indicate that EGF may regulate growth of
mammary epithelium: (a) EGF promotes growth of normal ro
dent mammary tissue and rodent breast cancer (25); (o) it
enhances growth of normal human mammary epithelial cells in
short-term culture (24); and (c) it stimulates mitosis of cells
derived from benign human breast fibroadenomas (23). The
effect of EGF on malignant human breast tissue has not been
reported in detail, but one might speculate that certain breast
cancers could retain the sensitivity to EGF observed with
nonmalignant mammary cells. In the present studies, we have
examined the effects of EGF on human breast cancer cells
maintained in long-term tissue culture, a system well suited for
biochemical studies of hormone action since the environmental
milieu of the cells can be carefully defined. This model system
has been characterized in detail and is used by several labo
ratories for studies of steroid and peptide hormones (18). We
find that EGF stimulates proliferation of a human breast cancer
cell line, suggesting that it may be an additional hormonal
factor regulating breast cancer growth in certain patients with
this disease.
MATERIALS AND METHODS
Materials. Mouse EGF was purchased from Collaborative
Research, Inc. (Waltham, Mass.). Porcine insulin (Lot 615D63-10, 25.4 units/mg) was the gift of Dr. M. Root, Lilly
Research Laboratories (Indianapolis, Ind.). Crystalline TCA was
purchased from Baker Chemical Co. (Phillipsburg, N. J.), and
[3H]thymidine (47 Ci/mmol), [5-3H]uridine (29 Ci/mmol), and
L-[U-'"C]leucine (342 mCi/mmol) were obtained from the Radiochemical Centre, Amersham, England.
Cells and Tissue Culture Techniques. Both human breast
cancer cell lines were initially derived from malignant effusions
of women with metastatic breast cancer and have been in
continuous tissue culture for at least 4 years. Characterization
of the human and mammary origin of these cells has been
summarized previously (10, 13, 18). The MCF-7 cell line was
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2361
C. K. Osborne et al.
provided by Dr. H. D. Soûleat the Michigan Cancer Foundation
(21). These cells are maintained in monolayer culture in IMEM
(Grand Island Biological Co., Grand Island, N. Y.) supple
mented with glutamine (0.6 g/liter), penicillin (62 mg/liter),
streptomycin (130 mg/liter), insulin (1.0 nw), and 5% calf
serum. The MDA-231 cell line was initially isolated by Dr. R.
Cailleau at M. D. Anderson Hospital and Tumor Institute (4) and
supplied to us by Dr. M. Lippman of the National Cancer
Institute. These cells are maintained in the same medium as
the MCF-7 cells but with 10% fetal bovine serum and no insulin
supplementation. Both cell lines are grown in a humidified
incubator in 5% CO? at 37°,and both are free of Mycoplasme
contamination. The cells are subcultured weekly with the use
of 0.05% trypsin:0.02% EDTA in 150 mw NaCI to remove cells
from the culture dish. In all experiments, cells were harvested
by suspending them with this same solution.
Precursor Incorporation and Growth Studies. Methods for
the measurement of precursor incorporation into macromolecules and cell growth have been summarized previously (17,
20). Briefly, incorporation studies were performed by plating
cells in replicate to a subconfluent density (about 7 x 10s
cells/well) in 4 ml of IMEM supplemented with the appropriate
serum in multiwell culture dishes (FB-4-TC; Linbro Chemical
Co., New Haven, Conn.). After the cells had attached to the
dish (24 hr), the medium was exchanged for serum-free IMEM.
After a further 24 hr, hormones (or hormone-free carrier for
controls) were added directly to the incubation medium, and
the cells were incubated for specified times. During the last 2
hr of incubation, cells were pulsed with labeled thymidine (0.5
fiCi/ml), uridine (1.0 to 2.0 /¿Ci/ml), or leucine (0.5 /nCi/ml)
and then were harvested. An aliquot of the cell suspension was
taken for DNA determination (3), and the remaining cells were
disrupted by sonicating for 3 sec in a Heat-Systems Ultrasonic
sonicator (Plainview, N. Y.) with a microtip at the lowest setting.
Precursor incorporation was determined by measuring the
radioactivity precipitated in 10% TCA after collection on Millipore filters (0.45 firn, type HA). The rates of precursor incor
poration were linear under the conditions used.
Cell growth studies were performed by determining the total
DNA (3) or protein (15) content of cells plated in replicate on
Petri dishes (Falcon Plastics, Oxnard, Calif.) after incubation in
serum-free medium (unless stated otherwise) with or without
EGF or other factors. Cell number was determined by visual
counts in a hemocytometer. Changes in DNA content closely
paralleled changes in population size.
TLI. The TLI was determined by a modification of previously
published methods (14). Cells were plated in replicate and
incubated with hormones, as described for the incorporation
studies. At the end of the incubation period, they were pulsed
for 1 hr with [3H]thymidine (5 juCi/ml), washed twice, harvested,
and centrifuged (600 rpm for 2 min). The supernatant contain
ing the harvesting medium was discarded. The cells were
resuspended in chilled (4°) Dulbecco's phosphate-buffered
saline (pH 7.4, without Ca2+ or Mg2+) at a concentration of
about 2.5 x 105 cells/ml. Two-tenths ml of the diluted cell
suspension was added to a cytocentrifuge cup and centrifuged
for 5 min at 1000 rpm (Shandon Elliott, Southern Instruments,
Inc., Sewickley, Pa.). After a drying time of 4 hr, the cytocentrifuge-prepared
slides were dipped 10 times in each of 2
staining dishes containing 5% cold TCA and then dipped 5
times in a dish containing methyl alcohol. After drying, the
2362
slides were dipped in Kodak NTB-2 emulsion, exposed for 24
hr in a light-tight box, developed with D19 developer, and fixed
with Kodak general purpose fixer. The slides were dried and
then stained with May-Grunwald-Giemsa.
A total of 500 cells
were counted for each of 4 slides representing one experimen
tal group (2000 cells total). The labeling index (labeled/unlabeled cells) was calculated by counting the fraction of intact
cells containing 8 or more grains over the nucleus. Background
counts for the autoradiographs were always less than 5 grains/
cell nucleus.
Statistical Analysis. When applicable, the means of experi
mental groups were compared by using Student's f test.
RESULTS
Effects of EGF on Growth. EGF significantly stimulates
growth of the MCF-7 human breast cancer cells (Chart 1 A and
B). Cell proliferation as determined by measuring the total DNA
and protein content of dishes plated in replicate was aug
mented by the addition of EGF (10 ng/ml) to cells growing in
serum-free medium. Saturation density of the cells was reached
by 7 days in both control and EGF-treated cultures. At this
time, DNA content was increased nearly 2-fold and total protein
was increased 3-fold in cultures incubated with the polypeptide. The increased DNA content in this experiment paral
leled a 2-fold increase in actual cell number after 5 days
[control = 7.2 ±0.2 x 10s (S.D.) cells/dish; EGF = 13.3 ±
0.9 x 105 cells/dish],
indicating that EGF stimulates cell
division and not just synthesis of DNA and protein. The obser
vation that EGF stimulates protein synthesis to a greater extent
than DNA synthesis and mitosis (a hypertrophie effect) was
reproducible in multiple experiments. In a separate but similar
experiment (Chart 1C), the effect of EGF on actual cell number
was determined. The initial doubling time was reduced from
about 36 to 24 hr by the hormone. It should be emphasized
that control cells in serum-free medium alone were not dying
under these conditions but continued to synthesize macromolecules and to divide. Thus, the EGF effect is not simply to delay
or prevent cell death but to increase multiplication of these
cells.
Growth of MCF-7 cells is extremely sensitive to EGF (Chart
2). Stimulation of DNA and protein synthesis was observed
with as little as 0.01 ng of EGF per ml (p < 0.05 and 0.005,
respectively). Half-maximal stimulation was noted with concen
trations between 0.1 and 1.0 ng/ml, and maximal stimulation
was noted at 10 ng/ml. These EGF concentrations are similar
to those required for a mitogenic effect in other tissues (5, 11).
Furthermore, these concentrations are within the range of
concentrations reported for EGF in human secretions and
plasma by radioimmunoassay (9, 22). A modest but consistent
reduction in the stimulatory effect of EGF was noted at con
centrations of 50 ng/ml or greater.
The effect of EGF on growth is evident even when MCF-7
cells are plated at a very low ("cloning") density (Table 1). In
this experiment, cells were plated at a density of 500 cells/60mm Petri dish and incubated for 15 days in serum-free medium
with and without EGF or 5% calf serum. Surprisingly, cells
maintained in serum-free medium alone slowly divided to reach
a saturation density of 11.4 x 10" cells. The addition of EGF
resulted in a 2-fold increase in saturation density. However, the
effect of EGF is only about 10% of that observed with calf
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Stimulation of Human Breast Cancer by EGF
1000-,
0
again, no EGF effect was observed (Table 2). Similar experi
ments using higher serum concentrations
(1%) or serum
stripped of steroid hormones and probably many proteins by
treatment with dextran-coated charcoal failed to produce EGF
stimulation of the MDA-231 cells (data not shown). Of interest
is our previous observation that these cells are also insensitive
to insulin (20). The factors in serum responsible for growth
stimulation of this cell line remain unknown.
Effects of EGF on Precursor Incorporation. We also ex
amined the effects of EGF on the rates of incorporation of
radioactive precursors into macromolecules in MCF-7 cells.
The sensitivity of these cells to physiological concentrations of
EGF was confirmed by the increased incorporation of [3H]thymidine, [3H]uridine, and [14C]leucine into TCA-precipitable
I
material (Chart 3). Slight stimulation was observed with 0.10
ng of EGF per ml, and maximal stimulation was observed with
10 ng/ml, in parallel with the dose-response curve of growth
stimulation (Chart 2). The magnitude of the maximal EGF
stimulation of precursor incorporation above control values (30
50 n
0
I
IOO
Chart 2. EGF dose response in MCF-7 cells Cells were plated as described
in Chart 1 (4 x 105 cells/100-mm
Petri dish), and EGF was added at the
indicated concentrations. The cells were fed fresh medium on Day 3 and har
vested on Day 5 for DMA and protein determination
Results are the means of
triplicates. Bars, S.E.
1
5357
Days
Chart 1. Effects of EGF on growth of MCF-7 cells Cells were plated in
replicate (1 x 10s cells/100-mm
Petri dish) as described in "Materials and
Methods." After 24 hr in serum-free medium, some dishes received EGF at 10
ng/ml (Day 0). Cells were fed with fresh medium with or without EGF on Days 2.
4, 6, 8, and 10. On the days indicated, cells were harvested for determination of
total protein in A (A. controls; A, EGF) or total DMA in e (•,controls; O. EGF). C.
a similar experiment in which the cell number was determined on the days
indicated (•.controls; O. EGF). Results are expressed as the means of triplicates.
Bars. S.E.
serum, indicating that other factors in serum are also important
for optimal growth of these cells.
In contrast to the effect on MCF-7 cells, EGF had no stimu
latory effect on DNA or protein synthesis in the MDA-231
human breast cancer cell line (Table 2); in fact, a slight de
crease in DNA and protein was observed with cells incubated
with EGF. Because an EGF effect on certain cells in culture
has been reported to require a cofactor present in low concen
trations of serum (11, 23), we also examined the effect of EGF
on these cells in the presence of calf serum (0.5%). Calf serum
itself significantly augmented macromolecular synthesis, but,
JULY
Table 1
Effect of EGF on growth of MCF- 7 cells plated at cloning density
Cells were plated as described in "Materials and Methods in medium con
taining 5% calf serum at a density of 500 cells/60-mm Petri dish. After the cells
had attached to the dish (24 hr). the medium was changed to serum-free medium
alone (control), serum-free medium plus EGF (10 ng/ml). or fresh medium plus
5% calf serum. EGF was added to appropriate cultures every other day. Spent
medium was exchanged for fresh medium with appropriate additions every 4
days Cells were harvested for cell counts, DMA, and protein determination on
Day 15.
of
10")11.4
cells (x
(fig)3.8
(/ig)55.9
Control
EGF
Calf serumNo.
±0.6a
20.2 ±0.7
229.4 ±5.0DNA
Mean ±S.E. of quadruplicate
±0.2
± 4.0
7.5 ±0.3
149.3 ± 6.4
85.8 ±1.4Protein 1091.4 ±60.1
determinations.
Table 2
Effect of EGF on growth of the MDA-231 cell line
Cells were plated as described in 'Materials and Methods" and in Chart 1.
On Day 0, cells were divided into 4 groups: control (serum-free medium); EGF
(10 ng/ml in serum-free medium); 0.5% calf serum; or 0.5% calf serum plus
EGF. Fresh medium was added on Day 3. and cells were harvested for DNA and
protein determination on Day 5.
Control
EGF
Calf serum
Calf serum + EGFDNA
0,g)76.2
±1.6a
69.2 ±2.3
133.6 ±1.5
128.0 ±1.1Protein
(/ig)1561
±34
1355 ±34
2587 ±170
2406 ±68
Mean ±S.E. of triplicate determinations.
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2363
C. K. Osborne et al.
to 100% depending on the experiment) was similar to the
maximal stimulation induced by insulin in these cells (17).
The time course of the effects of EGF on thymidine, uridine,
and leucine incorporation into macromolecules is also similar
to that observed with insulin (17). Stimulation of leucine and
uridine incorporation occurred shortly after EGF addition (by 3
hr) and was maximal from 12 to 18 hr (Chart 4). On the other
hand, EGF stimulation of thymidine incorporation was evident
only after a lag period of about 12 hr; maximal stimulation
occurred at about 24 hr incubation with EGF. This ordered,
sequential stimulation of precursor incorporation by EGF sug
gests that it is not simply due to altered precursor uptake into
the cell (usually an immediate effect) with subsequent changes
in the specific activity of the precursor pool. The fact that EGF
also stimulates the thymidine labeling index (see below) sup
ports that contention and suggests that an effect of EGF may
be to increase the proportion of cells actively synthesizing
DMA.
TLI. The TLI provides an estimate of the fraction of cells in a
population actively engaged in DMA synthesis at a given time
(14). EGF significantly augmented the TLI of MCF-7 cells (Table
3). Nearly a 2-fold increase in TLI above that seen in cells
maintained in control medium was observed for cells incubated
with EGF for 24 hr. This effect on the TLI was identical to that
80 n
O.I
IO
IO
EGF (nq/ml)
Chart 3. Effects of EGF concentration on precursor incorporation. Cells were
plated in replicate as described in "Materials and Methods.'
After 24 hr in
serum-free medium, EGF was added at the indicated concentrations for 20 hr.
During the last 2 hr. the cells were pulsed with [3H]thymidine (0.5 /iCi/ml),
[3H]uridine (2.0 /iCi/ml), or ["C]leucine (0.5 fiCi/ml). The radioactivity precipi
tatale by 10% TCA was determined and expressed as dpm/dish/2
hr and shown
here as a percentage above control. Results are means of triplicates. S.E.'s were
between 2 and 10%.
Chart 4. Time course of the effect of EGF on labeled precursor incorporation
into macromolecules by MCF-7 cells. Cells were plated in replicate as described
in "Materials and Methods." EGF (10 ng/ml) was added at Time 0. [3H]Thymidine
(0.5 fiCi/ml). [3H]uridine (2.0 /iCi/ml). or [MC]leucine (0.5 fiCi/ml) were added
for a 2-hr pulse just prior to harvesting at the times shown. Incorporation into
TCA-precipitable material was determined and expressed as dpm per /ig DNA
per 2 hr. Results are the means of triplicates or quadruplicates. Bars, S.E.
2364
Table 3
Effect of EGF and insulin on the TU of MCF- 7 cells
Cells were plated in replicate as described in Chart 1 and then incubated with
EGF (10 ng/ml) and/or insulin (10 nM) for 20 hr. The cells were pulsed with
[3H]thymidine (5 fiCi/ml) for 1 hr, and the percentage of cells with labeled nuclei
was determined by autoradiography as described in "Materials and Methods.
1b35±
ControlEGFInsulinEGF
236 ±
233 ±
±1
+ insulinTLIa20
Expressed as percentage of labeled nuclei.
6 Mean ±S.E.
seen with insulin (10 nw). Interestingly, no additive effect on
TLI was observed in cells incubated with optimal concentrations
of the 2 hormones together. Thus, a major effect of EGF and
insulin in these cells may be to increase the fraction of cells in
the population that synthesize DMA. Additional studies will be
required to more accurately define the effects of these hor
mones on other parameters such as cell cycle transit times.
Because the effects of EGF and insulin on the TLI were not
additive, we wondered whether cell proliferation was influenced
in a similar fashion by these 2 growth factors. When MCF-7
cells were incubated with optimal concentrations of EGF or
insulin for 6 days, the effect on cell growth as measured by
total DMA or protein content of dishes plated in replicate was
strikingly similar (Table 4). A 50% increase in DMA content and
a 75 to 100% increase in total protein were observed. However,
an additive effect on DMA and protein content was not observed
when the cells were incubated with both hormones. On the
contrary, a slight decrease in DMA was noted for cells exposed
to both EGF and insulin together, and only a minimal increase
(not additive) in total protein was noted in this group. These
data suggest the possibility that the stimulation of MCF-7 cells
by EGF and insulin may involve a common step in the mecha
nism of action of these hormones. Furthermore, they suggest
that the pathways of regulation of DMA and protein synthesis
by these hormones may be different, since changes in protein
content do not always parallel changes in DNA or cell number.
Influence of Glucocorticoids and Serum on the EGF Re
sponse. Several reports have indicated that the presence of
either serum or the glucocorticoid dexamethasone enhances
the mitogenic response of human fibroblasts in culture to EGF
(1,5, 11, 23). Presumably, serum provides a cofactor neces
sary for a maximum EGF response, whereas dexamethasone
may increase the binding of EGF to its receptor. In contrast,
the response of MCF-7 cells to EGF is not enhanced by
dexamethasone or by low concentrations of calf serum (Tables
5 and 6, respectively). Dexamethasone (1.0 or 100 nw) for 6
days resulted in a minimal increase in total cell protein or DNA
per dish (Table 5). However, when dexamethasone was added
with EGF, a decrease in protein and DNA compared to EGF
alone was observed (p < 0.025). This antagonism of the EGF
response by dexamethasone is similar to antagonism by dex
amethasone of the mitogenic response to insulin in MCF-7 (1 7)
and ZR 75-1 (19) human breast carcinoma cells.
Similarly, calf serum did not synergistically enhance the
effect of EGF in MCF-7 cells (Table 6). In serum-free medium
EGF caused a 3-fold rise in total protein per dish and a 70%
increase in DNA over untreated cells. Calf serum stimulated
DNA and protein accumulation in a dose-dependent manner
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Stimulation of Human Breast Cancer by EGF
Table 4
Effect of EGF and insulin on MCF-7 cell growth
Cells were plated as described in Chart 1 (2 x 10s cells/60-mm Retri dish)
and exposed to EGF (10 ng/ml) and/or insulin (1.0 nM) for 6 days with a media
change on Day 3.
Control
EGF
Insulin
Insulin + EGFDMA
(¿ig)33.5
/dish
±2A°
(fig)406
±24
805 ±27
49.6 ±0.7
728 ±20
50.4 ±2.5
44.5 ±1.4Protein/dish931 ±56
Mean ±S.E.
Table 5
Effect of dexamethasone on the MCF-7 response to EGF
Cells were plated as described in Chart 1 (5 x 10s cells/100-mm Petri dish).
EGF (10 ng/ml), dexamethasone, or both were added to the cells for 6 days.
Fresh media with hormones were exchanged for spent media on Day 3.
ControlEGFDexamethasone
nM)Dexamethasone
(1.0
nM)+
(1.0
EGFDexamethasone
nM)Dexamethasone
(100
MM)+
(100
EGFDNA/dish
s Mean
(fig)115
5a216
±
4133 ±
4185 ±
6137 ±
8164 ±
± 4Protein/dish
(jig)1709
±1134233
1252321
±
1763403±
1802405±
243518 ±
± 280
± S.E.
Table 6
Effect of serum on the MCF-7 response to EGF
After plating (1 x 105cells/100-mm Petri dish), cells were incubated with the
factors shown for 5 days prior to harvesting for protein and DMAdetermination.
Spent media were exchanged for fresh media on Day 3.
Control
EGF (10 ng/ml)
Calf serum (0.1%)
Calf serum (0.1%) + EGF
Calf serum (0.5%)
Calf87a
serum (0.5%) + EGFDNA/dish
Mean ±S.E.
(/ig)19.6
±1.5a
33.6
30.4
46.2
62.7
56.1
(^g)234
676
±0.5
513
±0.4
±0.8
965
±1.6
1082
1171
±1.6Protein/dish 326
±
±
±
±
±
43
30
17
23
±
but did not augment the EGF effect. In fact, in the presence of
0.5% calf serum EGF increased protein accumulation 30%
above that seen with the calf serum alone, but no additional
stimulation of DMA content was observed. Thus, serum factors
are not required for, nor do they synergistically enhance, the
mitogenic response to EGF of MCF-7 cells.
DISCUSSION
The importance of EGF for cellular growth regulation in vivo
has not been defined. In vitro studies, however, suggest that a
variety of tissues may be targets for EGF action. Mammary
epithelium is a potential target for this hormone. It is of interest
that levels of EGF in humans are higher in females and are
increased further by pregnancy or exogenous sex steroid
hormone administration (9), conditions which drastically alter
mammary gland growth and development. Furthermore, cell
culture studies suggest that EGF regulates growth of rodent
and human mammary epithelial cells (23-25). The present
studies demonstrate that EGF is also a potent growth stimulus
for human breast cancer cells in culture, thus confirming a
preliminary report that EGF is a necessary ingredient for growth
maintenance of these cells in a defined serum-free medium (2).
Physiological concentrations of EGF enhance MCF-7 cell mul
JULY
1980
tiplication and increase the rates of thymidine, uridine, and
leucine incorporation into macromolecules in these cells. A
major effect of this hormone appears to be an increase in the
number of S-phase cells in the population. Whether this is due
to recruitment of nonproliferating cells into the cell cycle or
changes in cell cycle transit times will require further study.
The observation that another breast cancer cell line, the MDA231, is not sensitive to EGF suggests that only certain breast
tumors retain the biomolecular machinery necessary for an
EGF effect and that the stimulatory effect of EGF on the MCF7 cells is not an artifact of tissue culture conditions.
Unlike effects on certain other types of cells in culture, the
effect of EGF on MCF-7 cells is evident in serum-free medium
and is not exaggerated in the presence of calf serum. In
addition, dexamethasone does not augment the EGF response
in these cells, but, in fact, slightly antagonizes the response.
These disparate results among different cells in culture may be
due to culture conditions, or may reflect genuine differences
among tissues in their hormonal responsiveness. We have also
observed that the mitogenic action of insulin is antagonized by
glucocorticoids in human breast cancer cells in tissue culture
(17, 19). One possible explanation for these effects is that
dexamethasone could cause a nonspecific alteration of the cell
membrane, thereby affecting both EGF and insulin receptors.
Another possibility is that dexamethasone opposes the action
of EGF and insulin at a common site distal to receptor binding.
The effects of EGF on the MCF-7 cell line are strikingly
similar to the effects we have reported previously for insulin
(1 7, 19). (a) The stimulation of growth and precursor incorpo
ration into macromolecules is quantitatively equivalent with
both hormones, (b) Both hormones stimulate cell proliferation
and the TLI, and the effects are not additive when the cells are
exposed to optimal concentrations of both hormones, (c) The
time course of stimulation of thymidine, uridine, and leucine
incorporation by insulin and EGF are nearly identical, (d) Glu
cocorticoids modulate the effects of both hormones in a similar
manner, (e) The MDA-231 cell line, which is unresponsive to
insulin, also fails to respond to EGF. These observations sug
gest that EGF and insulin may stimulate a common biochemical
pathway in MCF-7 cells leading to enhanced growth. Since
specific receptors for each of these polypeptides have been
detected in target cells and since neither hormone competes
for the receptor of the other in MCF-7 cells (20)," it is unlikely
that a "common pathway" involves a common receptor binding
site at the cell membrane. However, after initial binding of these
hormones to receptors dispersed on the cell surface, it has
been shown by fluorescent techniques that bound insulin and
EGF rapidly migrate on the membrane to form patches where
both are internalized within the same vesicle by a common
pathway (16). Whether patching and internalization are related
to the growth effects of these hormones is not known, but it is
interesting to speculate that the similar responses to EGF and
insulin observed in MCF-7 cells are related to this event.
In summary, we have shown that EGF stimulates growth of
human breast cancer cells in tissue culture. These results, in
concert with other studies of rodent and human mammary
epithelium, suggest the possibility that EGF has a regulatory
role in the growth and development of the breast and perhaps
of breast cancer.
' C. K. Osborne and B. Hamilton, unpublished data.
2365
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C. K. Osborne et al.
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CANCER
RESEARCH
VOL. 40
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Epidermal Growth Factor Stimulation of Human Breast Cancer
Cells in Culture
C. Kent Osborne, Barbara Hamilton, Grace Titus, et al.
Cancer Res 1980;40:2361-2366.
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