[CANCER RESEARCH 46, 2614-2618, May 1986]
Characterization of Growth Factors in Human Ovarian Carcinomas
Thomas Bauknecht, Marion Kiechle, Georg Bauer, and Jan W. Siebers
Department of (jynecology and Obstetrics, University of Freiburg. D-7800 Freiburg, Federal Republic of Germany
ABSTRACT
Kpidcrmal growth factor (EGF)-like factors with EGF competing and
cell growth stimulating activity were investigated in malignant and nonmalignant tissues. About 37% of ovarian carcinomas present an increased
factor activity between 9.0 and 19.3 ng EGF units/mg protein. In one
tumor 175.0 ng EGF units/mg protein were found. In extracts of nonmalignant tissues, the factor concentration was about 1.0-6.4 ng EGF
units/mg protein. Isoelectric focusing was performed to characterize these
factors. In normal ovaries and ovarian carcinomas, factors with EGF
competing activity focus at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9. In
ovarian carcinomas, an additional peak with EGF competing and cell
growth stimulating activity was found between pH 6.5 and 7.2. Similar
results could be achieved in other malignant tissues investigated. These
data indicate the presence of EGF-like factors. EGF itself focuses at pH
4.6 (G. Carpenter and S. Cohen, Annu. Rev. Biochem., 48: 193-216,
1979).
Specific EG K binding was determined in 12 ovarian carcinomas. In
five of these cases EGF receptors could be detected. In the EGF receptor
positive carcinomas, the content of EGF-like growth factors varied be
tween 0 and 9 ng EGF units/mg protein. In EGF receptor negative cases,
the content of EGF-like growth factors varied between 0 and 19.3 ng
EGF units/mg protein. The clinical data of 19 patients are also demon
strated.
INTRODUCTION
Ovarian cancer, with a 5-year survival rate of about 20-30%,
is the most fatal gynecological cancer (1). The mechanisms for
the induction of this cancer are not known. Woodruff (2)
suggested that the common ovarian cancers arising from the
germinal epithelium could be caused by agents introduced into
the peritoneal cavity through the natural pathway, i.e., vagina,
uterus, and fallopian tubes.
In vitro experiments have shown that agents such as tumor
promoters or viruses induce, after cell transformation, the syn
thesis of peptides with phenotypically transforming properties,
i.e., the TGFs1 (3). The action of TGFa and TGF/3 is mediated
by different membrane receptors, whereby the TGFas utilize
the receptor system of the EGF (4-7). The further pathway of
phenotypical cell transformation by these factors is unknown.
There are, however, some experimental data which suggest
that autonomous growth of transformed cells might be due to
a constitutive expression of growth factors and the membrane
receptor. The expression may be encoded by oncogenes or may
be under the control of oncogenes (8).
We have recently demonstrated the existence of EGF recep
tors in some ovarian carcinomas (9). It could be shown that the
EGF receptor status can be used as a prognostic factor (9, 10).
EGF receptor positive carcinomas were associated with a better
survival rate of the patients. It may be that spontaneously
originating tumors such as ovarian carcinomas produce EGFlike growth factors which result in blocking and down regulation
of the EGF receptors as described for other growth factors
produced by human tumor cells (8).
In this paper we report on EGF-like factors from normal and
Received 3/13/85: revised 12/30/85: accepted 1/30/86.
' The abbreviations used are: TGF, transforming growth factor; EGF. epider
mal growth factor: EBV, Epstein-Barr virus: EGF-RRA. epidermal growth factor
radiorcceptor assay: MEM. minimal essential medium; PDGF, platelet derived
growth factor; EA. EBV antigen.
malignant tissue extracts which are able to compete with EGF
available receptors. These extracts exhibited growth promoting
activities in 3T3 cells. Some tumors contain elevated levels of
EGF-like activity as compared with normal tissue.
MATERIALS AND METHODS
Highly purified EGF was purchased from Collaborative Research
(Waltham, MA), and carrier-free Na'25I was from Amersham/Buchler
(Braunschweig, Federal Republic of Germany). Cellulose acetate filters
(EGWP) were obtained from Millipore (Millipore Corp., Bedford,
MA). Ampholine solutions and Ultradex were purchased from LKB
(Uppsala, Sweden). All chemicals and reagents used were of analytical
grade.
Preparation of Tissues for Binding Assays with EGF. Tissues of
ovarian carcinomas, mammary carcinomas, malignant melanoma, sar
comas, normal ovaries, and myometrium were obtained at surgery and
kept at —90°C
until processed. The tissues were pulverized with a
dismembralo!", dissolved in 0.25 M sucrose (1:10, w/v), and centrifugea
at 100 x g for 10 min to remove unbroken material. After a further
centrifugation step for the supernatant (5000 x g, 30 min, 4°C),the
pellet was redissolved in 50 mM Tris-HCl (pH 7.3)-1 ITIMMgCl2-100
mM NaCl at a concentration of 1-2 ml/mg protein. The placenta!
tissues for the EGF radioreceptor assay were obtained after deliveries
and prepared by the same procedure.
Extraction Procedure. Pulverized tissues were extracted with l N
acetic acid (1:10, w/v) at 4°Cfor 4 h. After centrifugation (100,000 x
g, 30 min, 4°C),supernatants were dialyzed against distilled water
containing 1% glycerol or 50 mM Tris-HCl (pH 7.5) for 48 h (Spectrapor tubing; molecular weight cutoff, 3,500; Spectrum Medical Indus
tries, Los Angeles, CA). The materials were centrifuged to clarity,
tested for EGF competing activity, for cell growth promotion of 3T3cells, and for EBV antigen induction in Raji cells; and analyzed by
isoelectric focusing.
EGF-RRA. EGF was iodinated by the chloramine-T method, which
resulted in a specific activity of 2-3 x IO5cpm/ng. Binding of'"I-EGF
was performed on placenta! plasma membranes at a membrane concen
tration reaching approximately 5000 cpm specific binding in the EGFRRA. Placenta! plasma membranes (20 >il)were suspended in a total
volume of 200 ^1of binding buffer (50 mM Tris-HCl-0.1 % bovine serum
albumin, pH 7.3) containing 0.4 ng 125I-EGF and increasing amounts
of imlabeled EGF (2.5-100 ng). The incubation was carried out in
duplicate for 45 min at 37°C.After incubation the reaction was stopped
with 2 ml ice-cold Tris buffer, and unbound ' "I-EGF was removed by
Millipore filters saturated with 4% bovine serum albumin and washed
with 10 ml Tris buffer. The retained radioactivity was determined in a
gamma spectrometer. Total binding refers to the cpm bound in the
absence of unlabeled factor. Nonspecific binding refers to the counts in
the presence of 100 ng unlabeled EGF. Specific binding is obtained by
subtracting the nonspecific binding from the total bound radioactivity.
The ratio of bound radioactivity to free EGF was plotted as a function
of the percentage of displacement. To determine tissue EGF competing
activity, 100 »il
of a tissue extract previously dialyzed against binding
buffer were used instead of unlabeled EGF. The percentage of displace
ment of 125I-EGFwas determined as EGF units in ng. The EGF-RRA
was sensitive for 2 ng EGF units; the 50% intercept precision was 10
ng. For the estimation of EGF receptors in tumor tissues, 100 M'tumor
homogenate were used and the binding assay was performed in the
same procedure. The binding data were analyzed by Scatchard plot
(11).
Isoelectric Focusing. Dialyzed tissue extracts (about 500-800 p\)
containing 1% ampholine (pH 3-10) were applied to small columns
(0.6 x 12 cm), which were packed with a 1% ampholine-Ultradex
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GROWTH
FACTORS IN OVARIAN CANCER
suspension. Isoelectric focusing was carried out at 4°Cwith a constant
voltage of 170 V for about 1500 volt-h, followed by 400 V for 30 min
with the phosphoric acid (0.2%, v/v)-ethanolamine (0.4%, v/v) system.
After focusing, the gels were sectioned in 0.25-ml fractions and diluted
with distilled water to 0.5 ml. After pH determination and centrifugation, the supernatants were dialyzed at 4°Cagainst the binding buffer.
Each fraction was tested for competition of EGF binding, for cell
growth promoting activity and, occasionally, for EBV induction activ
ity.
Assay for Growth Promoting Activity. Semiconfluent cultures of NIH
3T3 cells were trypsinized, suspended in MEM plus 5% serum, and
washed in serum free MEM. Cells were seeded at 1 x IO4 per assay
(Costar 24 tissue culture clusters) in the presence of 1.5% platelet
depleted human plasma. After addition of the extracts, assays were run
for the time indicated (12). The cell numbers were determined under a
microscope in a Fuchs-Rosenthal chamber after trypsinization. Human
plasma (1.5%) plus saturating amounts of PDGF induced the same
proliferation rate as 5% fetal calf serum.
Preparation of Platelet Depleted Human Plasma. Heparinized blood
was drawn from a 37-year-old healthy male volunteer and was then
diluted with an equal volume of serum free MEM and centrifuged for
50 min at 500 x g and 4°C.The supernatant was centrifuged under the
same conditions and then finally for 60 min at 2000 x g. This super
natant was incubated for 30 min at 56"C, and the precipitate was
removed by centrifugation at 4500 x g for 60 min.
Other Procedures. Protein was determined by the dye fixation method
(Bio-Rad). Purification of EBV inducing factors has been described
recently (13). Culture of lymphoid cells (Raji) and the test for EBV
inducing activity were performed as described elsewhere (13).
RESULTS
100
) UNLABELED
EGF
Fig. 1. EGF radioreceptor assay. Percentage of displacement of 125I-EGF by
unlabeled factor or 100 n\ dialyzed tissue extracts compared with the control,
exposed to labeled EGF alone. Extracts of normal ovaries (
,
): tissue
extract of an ovarian carcinoma (
) with 14 ng EGF units/80 itg protein (175
units/mg).
The content of EGF-like factors was estimated in 19 different
ovarian carcinomas, 1 mammary carcinoma, 2 sarcomas, 2
endometrial carcinomas, 1 malignant melanoma, 6 normal
ovaries, 4 fibromyomas, and 8 normal myometria. Fig. 1 shows
the EGF competition on placental membranes by unlabeled
EGF or by acid extracts of normal ovaries and an ovarian
carcinoma by EGF radioreceptor assay. The percentage of EGF
competition and the protein content in 100 /¿I
dialyzed tissue
extract were measured and expressed as ng EGF units/mg
protein. Fig. 2 shows the results of activities measured in the
acid extracts of the tumors and nonmalignant tissues. Nonmalignant tissue extracts exhibit a factor concentration of about
1.0-6.4 ng EGF units/mg protein. A similar activity was found
in 12 ovarian carcinomas, 1 mammary carcinoma, 2 endome
trial carcinomas, and 1 sarcoma. In 3 ovarian carcinoma ex
tracts, no EGF-like activity was detectable. However, 6 ovarian
carcinoma extracts revealed elevated levels of EGF competing
activity between 9.0 and 19.3 ng EGF units/mg protein. In one
200-j
190180170160-
MELANOMA
20n
o
CE:
tt.
15-
Fig. 2. Content of EGF competing activity
in extracts of malignant and nonmalignant
tissues.
ADENOSARCOMA
10-
o
z
5o
u
ENDOMETRIUM CA
ENDOMETRIUM-CA 1
SARCOMA
BREAST CAOVARIAN
CARCINOMAS
OTHER
CARCINOMAS
NORMAL
OVARY
MYOMA
MYOMETRIUM
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GROWTH
FACTORS IN OVARIAN CANCER
ovarian carcinoma, 175.0 ng EGF units/mg protein were found.
The melanoma extract contained 200.0 ng EGF units/mg pro
tein. To clarify whether tissue extracts contain different factors
with EGF competing and cell growth stimulating activities, the
extracts were subjected to semipreparative isoelectric focusing.
A representative isoelectric focusing pattern is shown in Fig. 3.
The EGF competing activities comigrate with cell growth stim
ulating activity at pH 7.4, which is separated from the EA
inducing activity at pH 5.7. A PDGF analogous novel growth
factor from platelets has been described recently (12). The factor
20 —
-loo -
100 —
-
L
5
I—0
,CTION
NUMBER
20
FRACTION
NUMBER
Fig. 3. Isoelectric focusing of a tissue extract of a sarcoma (Table 2, Patient
30) with regard to inhibition of 12!I-EGF binding, cell growth stimulating activity,
and EBV antigen positive cells. The total amount of EGF competing activity
applied to the column was about 200 ng EGF units. After focusing. 100 M' of
each fraction were used for the determination of EGF competing and cell growth
stimulating activity and EA induction.
Fig. 4. Isoelectric focusing of tissue extracts of normal ovaries with regard to
inhibition of '"I-EGF binding, cell growth stimulating activity and protein
content of each fraction. The amount of EGF competing activity applied to the
column was about 50 ng (A) and 60 ng (B) EGF units.
Fig. 5. Isoelectric focusing of tissue extracts of different ovarian carcinomas
with regard to inhibition of '"I-EGF binding, cell growth stimulating activity,
and protein content of each fraction. The amount of EGF competing activity
applied to the column was about 20 ng (A) and 40 ng (B) EGF units. A, Patient
13; B, Patient 5 in Table 3.
can be easily monitored by its unique ability to induce latent
EBV antigen synthesis in lymphoid cells (12). A further EGF
competing activity is seen at pH 4.4, at which point the EA
induction and cell growth stimulating activity are slightly in
creased. 125I-EGF alone focuses at pH 4.6. These experiments
were further projected to compare the isoelectric points of
factors originating from 10 different ovarian carcinomas, 1
mammary carcinoma, 1 endometrial carcinoma, 2 sarcomas, 1
melanoma, and 2 normal ovaries.
The profiles from the normal ovarian extracts are shown in
Fig. 4. The ovarian derived factors focus at pH 6.0-6.2 and 4.5,
comigrating with cell growth stimulating activity. An additional
peak with EGF competing activity was detected at pH 8.1-8.4,
which is clearly separated from the pH 6.0 peak by a flat pH
gradient (Fig. 4).
When the same experiments were performed with tissue
extracts from ovarian carcinomas, the EGF competing activity
peaks appeared at pH 8.0-8.7, pH 5.9, and pH 3.9 (Fig. 5). In
contrast to the normal ovarian extracts, an additional peak of
EGF competing and cell growth stimulating activity was de
tected at pH 6.8. This material (pH 6.8) has no EA inducing
activity (data not shown). The isoelectric points of EGF com
peting and cell growth stimulating activity of the other investi
gated ovarian carcinomas are listed in Table 1. Nine of 10
extracts investigated revealed factor activity at pH 6.5-7.2.
Factor activity at pH 7.6-9.0 was detected in 7 of 10 carcino
mas, at pH 5.7-6.3 in 4 carcinomas, and at pH 3.6-4.9 in 5
carcinomas. The results for other extracted tumors and 2 nor
mal ovaries are listed in Table 2.
Table 3 presents the clinical data, the EGF receptor status,
and the amount of EGF competing activity from the investi-
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GROWTH
FACTORS
IN OVARIAN
gated ovarian carcinomas. In 12 cases the EGF receptor status
was determined. Five of 12 ovarian carcinomas were EGF
receptor positive. These tumors contain less factor activity than
most receptor negative ovarian carcinomas (Table 3). The low
CANCER
number of cases suggests that tumors not responding to chem
otherapy tend to contain higher amounts of EGF-like factors.
RESULTS AND DISCUSSION
The results of this study indicate that malignant and nonmalignant
tissues contain factors competing for available EGF
Isoelectric
points
of
EGF
competing
activity
and
growth
stimulating
Table
activity from different ovarian carcinoma extracts
receptors. The crude tissue extracts tested in cell culture show
cell growth stimulation of arrested untransformed 3T3 cells
which cannot be induced by native EGF (data not shown). It
'"!-EGFbinding(top
could be demonstrated that some tumors contain or produce
EGFcompetingactivity/column(ng)20403060205020505070EGFcompetingactivity(ng
ofgrowthstimula elevated levels of EGF-like factors. About 37% of all investi
EGFcompet
EGFunits/mgprotein)0.30.8193.6122.80.317.26.3175Isoelectricpoints
ofeachpeak)2858254441403490453420202048205053309090904040404661Total
gated ovarian carcinomas present increased factor activity com
ingactivity6.96.37.76.64.27.6-7.26.88.9-8.56.85.99.08.54.9-3.66.64.38.06.53.68.47.26.1-5.86.84.28.27.05.7%ofinhibitionof
tion7.96.86.6ND"8.86.8-5.9ND6.64.3NDNDNDND
pared with nonmalignant tissues. Several reports describe
Patient45810111213141619Isoelectricpointsof
growth factors with transforming activity in serum. Platelets,
in particular, produce TGFa, TGF0, PDGF, and a novel plate
let derived growth factor (12-14). Therefore it can be assumed
that the factor activity found in tissue extracts is composed of
several factors, some of which originate from serum or platelets.
Isoelectric focusing experiments revealed EGF-like factor
activity at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9 using
nonmalignant and malignant tissue extracts. EGF itself is char
acterized by an isoelectric point of 4.6 (15). Factor activity from
tissue extracts with an isoelectric point of 4.5 probably repre
sents endogenous EGF. An additional peak with EGF compet
ing and cell growth stimulating activity was detected in malig
nant tissue extracts with an isoelectric point of 6.5-7.2.
Isoelectric focusing of a sarcoma extract demonstrates a
different migration of EGF competing and cell growth stimu
lating activity (isoelectric point, 7.4) compared with the EA
inducing activity (isoelectric point, 5.7), which probably repre
sents endogenous EBV inducing factor from platelets (12).
Similar results could be achieved with extracts of ovarian car
cinomas. We previously reported that EGF receptors can be
found in about 30% of ovarian carcinomas, a result which
correlates with the clinical follow-up of the patients (9, 10). It
seems possible that EGF receptor negative carcinomas contain
more EGF-like factors that possibly mask the EGF receptors.
This assumption is compatible with the finding that tumors
with increased EGF-like activity tend to respond poorly to
1ND. not done.
chemotherapy.
Table 2 Isoelectric points of EGF competing activity and growth stimulating activity from different malignant and nonmalignant tissue extracts
points of
EGF competing
activity8.1
Patient
Tissue20
ovary22
of inhibition of
'"I-EGF binding
(top of each
peak)47
EGF
competing
activity/column
(ng)SO60EGF
competing
activity
points of
(ng EGF units/mg
growth
protein)3.02.7Isoelectric
stimulation6.2
Normal
6.0
4.58.46.0
60
7025
4.4%
40
40Total
4.5ND°
Normal ovaryIsoelectric
carcinomaMammary
2627282930Endometrial
carcinomaSarcomaMelanomaSarcoma4.1-3.25.84.4-4.07.54.68.6-8.24.2-3.27.45.74.440807060957575301240308040602002.20.71.1200.015.0NDND8.25.74.48.65.74.37.45.74.4"
ND, not done.
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GROWTH
FACTORS IN OVARIAN CANCER
Table 3 EGF receptor status and ECF competing activity in ovarian carcinomas compared with clinical data of the patients
receptorResponseYesYesYesYesYesYesMixedNoNoNoNoNoNoNoNoNoNEDNEDfiâ„¢Â
TumorPatient1234567891011121314IS16171819202122232425StageIIIIVIIIIIIIIIIIIIIIIIIIIIIIIIIIIVIIIIIIIVIIIIVlibIGrading22313323223211TypeClear
competing
activity
(ng EGF units/
M)Neg.Neg.125ND1.238.2Neg.Neg.Neg.NDNDNDNDNeg.NDNeg.5NDNormal
10-'°
protein)Neg.Neg.5113ND690Neg.Neg.Neg.NDNDNDNDNeg.NDNeg.1
mgprotein)6.03.0Neg.0.30.89.0Neg
cellSerousSerousSerousSerousSerousUndifferentiatedSerousSerousSerousSerousSerousSerousMucinoidClear
cellSerousUndifferentiatedMucinoidMucinoidSurgeryS(>2)S(>5)S(>2)S(>2)S(<2)S(<2)S(>2)S(>2)S(>2)S(>2)S(<2)S(>2)S(<2)S(<2)S(<1)SSSS(<I)T
+CEGF
(perimenopausal)Normal
ovary
(perimenopausal)Normal
ovary
(perimenopausal)Normal
ovary
(postmenopausal)Normal
ovary
(postmenopausal)Normal
ovary
ovary (premenopausal)EGF
»,binding capacity; S, surgery (tumor rest in cm); P, c£s-platinum;A, Adriamycin; C, cyclophosphamide; ND, not done; NED, no evidence of disease; Neg.,
negative.
The exact nature of the EGF competing activity of tumor
extracts remains to be defined. The preliminary estimation of
the molecular weight of the factors from some tumors indicated
a high molecular weight of 30,000-60,000. Sherwin et al. (16)
found high molecular weight TGF activity with EGF competing
activity in the urine of patients with disseminated cancers.
Kaplan and Ozanne (17) have shown that the transforming
activity strongly depends on the cell system used. Thus the
EGF competing activity found in the present study could be
identical to that described by Sherwin et al. (16).
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2618
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Characterization of Growth Factors in Human Ovarian
Carcinomas
Thomas Bauknecht, Marion Kiechle, Georg Bauer, et al.
Cancer Res 1986;46:2614-2618.
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