[CANCER RESEARCH 42. 5060-5066, December 1982]
0008-5472/82/0042-0000$02.00
Response to Estrogen by the Human Mammary Carcinoma Cell Line CAMA-11
Benjamin S. Leung,2 Shehla Qureshi, and Jonas S. Leung
Department of Obstetrics and Gynecology.
University of Minnesota Medical School, Minneapolis,
ABSTRACT
CAMA-1 cells, isolated from malignant pleural effusion, are
grown in long-term cultures as monolayers. The rate of growth
is dependent upon fetal bovine serum and estradiol. These
cells also exhibit a dose response to added 17/?-estradiol with
respect to the incorporation of radiolabeled thymidine into
cells. The uptake is increased at low levels of estradiol and
decreased at pharmacological levels of estradiol. The uptake
of uridine and leucine is also stimulated by estradiol in a doserelated manner. Induction of precursor uptake is not observable
until cells have been exposed to estradiol for approximately 10
hr or longer. Cells plated for different periods in steroidstripped serum remain sensitive to estrogenic stimulation and
show similar lag time in response. Estrogenic effect is not
noticeable in the absence of serum. Addition of prolactin can
partially restore estrogenic stimulation of thymidine uptake in
serum-free medium. Like other estrogen target tissues, these
cells contain cytoplasmic and nuclear estrogen receptors.
These results demonstrate that the CAMA-1 cell line is estrogen
dependent and that these cells may provide a promising model
for the in vitro investigation of the mode of estrogen action in
human breast cancer.
INTRODUCTION
It is known that the growth of breast cancer cells is influenced
by many hormones (13). Estrogen and prolactin are important
in stimulating growth of mammary tumors in rodents (2, 3, 11,
15,19, 22, 27). Recent reports (16-18, 21) demonstrated that
the growth of an established cell line (MCF-7, isolated from
pleural fluid of a breast cancer patient) was stimulated by low
levels of estrogen and inhibited by antiestrogen or high levels
of estrogen.
In this report, we present results of another established cell
line (CAMA-1 ) which responds to estrogen in precursor uptake
and cell growth.
MATERIALS
AND METHODS
Cells and Tissue Culture.
CAMA-1 is a human breast cancer cell
line originating from the malignant pleural effusion of a postmenopausal
woman with adenocarcinoma of the breast (4). These cells were grown
in T-75 flasks as monolayer cultures in Eagle's minimum essential
medium with Earle's salts and supplemented with 2 HIM L-glutamine,
100 units of penicillin per ml, 100 /ig of streptomycin per ml, and 15 or
25% FBS3 (Grand Island Biological Co., Grand Island, N. Y.). Culture
' Supported
in part by National Cancer Institute Research Grant 1R01-CA
25998. Part of this work was presented at the 60th Annual Meeting of the
Endocrine Society, 1978 (14).
2 To whom requests for reprints should be addressed, at Box 395, Mayo
Memorial Building, 420 Delaware Street S.E.. Minneapolis, Minn. 55455.
3 The abbreviations used are: FBS. fetal bovine serum; DCFBS. dextrancoated charcoal-treated fetal bovine serum; dThd. [3H]thymidine; ER, estrogen
receptor.
Received August 31,1981
5060
; accepted August 5. 1982.
Minnesota 55455
conditions, harvesting of cells for experiments, and seeding new pas
sages have been described previously (30).
Precursor Incorporation.
Cells growing in the log phase were plated
in T-25 flasks at a density of 5 x 10" cells/flask. They were grown in
minimum essential medium, supplemented with predescribed constit
uents and the corresponding proportions of FBS or DCFBS [serum
treated with dextran-coated charcoal to remove free steroids (30)]. In
experiments in which the effect of 17/?-estradiol was studied, it was
dissolved in ethanol before
ethanol concentration
was
intended period of time, cells
fiC'i), usually conducted for
addition to the medium so that the final
0.1% or less. After incubating for the
were pulsed with tritiated precursors (0.5
2 hr, unless otherwise stated. Following
aspiration of culture medium, cells were harvested by washing with icecold 0.9% NaCI solution, suspending on 0.025% trypsin-EDTA, and
collecting on glass fiber filters (Reeve Angel 934 AH) with a 22-hole
vacuum-manifold. After 2 or more washes with cold 0.9% NaCI solution,
the filters containing cells were put into scintillation vials. The cells
were digested by Protosol (New England Nuclear, Boston, Mass.) at
60° for 0.5 hr, and 10 ml Omnifluor-toluene
(New England Nuclear)
were added. Radioactivity was determined by a Beckman 350 scintil
lation system with an efficiency of 35 to 40% for tritium.
RESULTS
When CAMA-1 cells were cultured in a system not supple
mented with serum, cell proliferation and monolayer formation
were very poor, probably because there are abundant nutrients
and constituents in the serum that are essential for cell growth.
These substances include various steroid and peptide hor
mones. As shown in Chart 1, the serum requirement for cell
growth is evident. In the presence of estradiol (1 nw) and
different proportions of FBS, the initial rate of cell proliferation
among these cultures was not different (Chart 1A). However,
the total number of cells harvested on Day 7 from flasks with
10% or less FBS was much lower than with higher amounts.
This effect of serum was amplified on Day 10, because cells
grown in less than 20% FBS went into stationary phase by Day
7, contributing to a much lower cell yield at harvest than when
cells were grown in 25% FBS. These results show that a
depletion of essential nutrients from low serum cultures oc
curred following the fourth day of plating. When cells were
grown in serum treated with dextran-coated charcoal to remove
free steroids and other materials that adsorbed to the charcoal
surface, the rate of cell proliferation was much lower (Chart
1B). With the addition of estrogen, the rate of cell growth was
partially restored. This effect of estradiol was noticeable with
other proportions of DCFBS added (result not shown). Appar
ently, other constituents of serum that were removed by dex
tran-coated charcoal were also important for sustaining cell
growth.
To further test the requirement of estradiol for cell prolifera
tion, CAMA-1 cells were cultured in medium supplemented
with 25% DCFBS (Chart 2). While cell yield in 25% FBS
remained fairly constant at the end of each 7-day culture, from
2 separate experiments, cells grown in 25% DCFBS showed a
constant and continuous decrease in cell number at harvest. It
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Estrogen Response in Breast Cancer CAMA-1 Cells
10'
n
g
¿
« io'
10-
is*
24
10
DAYS
AFTER
PLATING
Chart 1. A. effect of FBS on the rate of CAMA-1 cell growth in the presence
of 1 nM estradiol and different amounts of FBS (as percentages). Points, close
means of triplicate determinations. B, cell growth in 15% DCFBS with (•)or
24
7
DAYS AFTER PLATING
OCFtS
10
without (O) 1.0 nw estradici (E). Growth curve in 15% FBS was taken from data
in A as a reference. Each value is the mean of triplicate determinations.
200E
15°QUl>
E
<ñ
•¿;•••131•S1-T-iISS;Ii;"-:
100_i—i
tu0
50nA——--1.'•\'
12345
104
4
6
PASSAGES
Chart 2. Effect of DCFBS on the maintenance of cell growth in culture. Each
culture was plated with 106 cells in a T-75 flask, as described in "Materials and
Methods," either in 25% FBS or 25% DCFBS without the addition of estradiol.
During harvest on Day 7, cells were suspended in 5 ml medium; 1-ml aliquot was
used for determination of cell number, another aliquot was plated as a new
passage. Data points (O) and (•)for DCFBS cultures were from 2 separate
experiments; •¿
and * values were from the same experiment. Cell numbers are
means of triplicates determined during harvest.
was observed that many viable, unattached cells were floating
in the media during prolonged culture in DCFBS. These float
ers, however, accounted for only a fraction of the cell loss. It
is known that complete removal of serum estradiol by dextrancoated charcoal adsorption is seldom complete; nevertheless,
the unadsorbed estradiol concentration must be too low for the
maintenance of cell growth following the first passage. In order
to assure that the removal of estradiol at least in part contrib
uted to cell death, an estrogen rescue experiment was con
ducted (Chart 3). To minimize the effect of any remnant estra
diol in the serum, a 10% DCFBS was selected for continuous
passages of the cells, either in the presence or absence of 1
nw estradiol. Cells grown in DCFBS, with or without estradiol,
stopped proliferating after the first passage. A 40% decline in
cell number was noted following the second passage in 10%
DCFBS without estradiol (Chart 3A). Although estrogen could
DECEMBER
1982
12345
WEEKLY PASSAGE
Chart 3. Effect of estradiol on cell proliferation in 10% DCFBS. CAMA-1 cells
were cultured in T-25 flasks, and on Day 7, cell numbers were determined and
were expressed as a percentage of the number of cells plated (50,000 cells,
100%). A. no estradiol added during plating for each new passage (open bar). At
the beginning of the fourth passage, cells from cultures without estradiol were
supplemented with 1.0 nM estradiol (stippled bar). B, 1.0 nM estradiol added at
time of plating of each new passage (hatched bar). Each value is the close mean
of triplicate determinations.
not stimulate cell proliferation in the second passage (Chart
38), it prevented the decline in cell number, and a partial
restoration of cell growth was noted by the fourth passage. If
estrogen was added at the beginning of the fourth passage to
chronically estrogen-deprived cells (Chart 3/4), a prompt dou
bling of cell number was noted. These results demonstrate that
estrogen is one of the basic elements necessary for CAMA-1
cell maintenance and growth in culture.
It is desirable to have a simple assay which can reflect the
incorporation of thymidine into DMA molecules. First, the meth
ods of dThd uptake into whole cells was compared to the
conventional method of 10% trichloroacetic acid precipitation.
Following pulsing for 0.25 to 4 hr (Chart 4/4), radioactivity
uptake into whole cells parallels that of trichloroacetic acidinsoluble materials. These 2 methods were further evaluated in
another experiment by determining the effect of estradiol upon
dThd uptake (Chart 46). Similar to results shown in Chart 4X\,
whether in the presence or the absence of estradiol, radioac-
5061
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
ß.S. Leung eÃ-al.
Chart 4. A, whole-cell dThd uptake versus trichloroacetic acid precipitation. Replicate nonconfluent monolayer cultures of CAMA-1 cells were labeled by adding
0.25 (iCi dThd to plates for the indicated time. Cultures
were terminated by removal of the labeled medium, one
rinse with 0.9% NaCI solution, and detachment with trypsin. O, cells filtered, dried, and counted in Budgetsolve
(RPI). A, cells filtered, dried, digested with H2O and Protosol (New England Nuclear), and counted in Omnifluortoluene (New England Nuclear). A, cells were precipitated
with 10% trichloroacetic acid and washed twice, and the
precipitates were digested with Protosol and counted in
Omnifluor-toluene. In all cases, dThd uptake is linear with
a slope of 1.0. B. effect of estradici on whole-cell dThd
uptake versus trichloroacetic acid precipitation. Replicate
nonconfluent monolayer cultures of CAMA-1 cells growing
for 48 hr either in the presence of estradici (1.0 nw) or in
its absence. Cells were pulse labeled with 0.25 (iCi dThd
for the time indicated and were harvested as described in
A, one set was filtered, dried, and counted in Budgetsolve
(•),while the other set was precipitated by trichloroacetic
acid (A). Estradiol-induced dThd uptake was expressed
as a percentage over controls without estradici (100%).
y''/s/-y/Ã '
10sLU(-
<_1
160D0Q
////"—
'^*^^^J^-**^~
„¿"~-^.—_-
—¿
-=5r——¿
O.SCLû10'IO3AE
"//r
12°zIO
///:
v//#
800OCOT
CELLTCA0.25
•¿
40LUB--•yv.
1234INCUBATION
0.5
1.0
tivity recovered by either method increased linearly from 1 to
4 hr in a double-logarithmic
plot (result not shown). The per
centages of estradiol-induced dThd incorporation derived from
these 2 methods were virtually the same (Chart 48). There was
a slight discrepancy at 0.25-hr pulsing. This is not unexpected
since many cellular events that lead eventually to DNA synthe
sis must first transpire before the actual incorporation of dThd
into DNA molecules. Presumably, a steady state of incorpora
tion was reached following 1 hr of pulsing, even in the presence
of estradiol.
The relationship between cellular dThd uptake and cell pro
liferation during a 7-day culture period was evaluated (Chart
5). The level of dThd uptake into whole cells again parallels the
cell yield derived from Coulter counting. Collectively, these
results show that 2-hr pulse labeling of whole cells can be used
as a reliable index for determination of dThd incorporation into
DNA molecules.
The duration required for estrogen to exert an effect on dThd
uptake was evaluated (Chart 6). In this experiment, CAMA-1
cells were incubated for 24 hr in 25% DCFBS prior to the
addition of estradiol. A slight decline of dThd uptake, probably
due to cell death or progression of cells from S phase to G2
phase, was observed during the first 8 hr of estrogen exposure.
By 16 hr, a more than 2-fold increase in dThd uptake was
reached, and remained at this maximal level up to 24 hr
following the addition of estrogen. When cells were cultured
for 48 hr in the presence of estradiol, added during plating, the
level of dThd was no different than the maximal level induced
by estradiol after a 16-hr exposure. In contrast, control cells
incubated for 48 hr without added estradiol had dThd radio
activity equivalent to uptake in cells exposed to estradiol for 2
hr. These results show that estradiol has an early effect on
CAMA-1 cells in the uptake of dThd. The observed lag time in
this experiment may represent the duration of the Gìphase
under estrogen environment. It appears that the transition of S
phase to G2 occurred from 8 to 16 hr following estrogen
exposure.
To rule out the possibility that the observed estrogen stimu
lation may be an artifact, the sensitivity of cells to estrogen was
examined in DCFBS following plating for 24 hr, 48 hr, or 7
days (Chart 7). In each case, estradiol induced a marked
increase in dThd uptake, and the induction invariably com
menced at 10 to 12 hr following the addition of estradiol.
5062
200JSï
2.0
4.0
WITH dThd
(log hr)
INCUBATION
104
WITH dThd (hr)
O
105
So
5 3J
" O
O
o î
*
•¿o
l
X
104
103
6
8
DAYS IN CULTURE
Chart 5. Growth rate of CAMA-1 cells in 25% FBS and 1 nM estradiol added
during plating. Harvesting of cells for Coulter counting and 2-hr dThd pulsing
were according to description in "Materials and Methods." Each value is the
close mean of triplicate determinations.
However, the pattern of the induction rate in each case was
not the same. Control cells without added estradiol uniformly
showed a much reduced rate of dThd uptake; only slight
increase was noted during the period between 10 and 16 hr
following the addition of vehicle as compared with estradioltreated cells. Under the present culture condition, cell number
at Day 7 was approximately double that at Day 2. A 20-fold
increase in dThd uptake in both control and estradiol-treated
cultures in comparison to respective cultures at 24 and 48 hr
suggests that most of the cells at Day 7 were in the G, phase.
The addition of dThd was able to initiate a traverse of the cell
cycle. This phenomenon was later on confirmed by cell cycle
analysis. Virtually no S-phase population remained on Day 7
while 80 to 90% of the cells were in the G, phase. The
stimulation of dThd uptake in estrogen-treated cells may then
represent a faster transition from Gìto S phase. These results
further indicate that estradiol is needed to promote a faster
progression from Gìto S phase in DCFBS environment.
In view of the fact that the estrogen-induced dThd uptake
might also be a reflection of the change in the pool size of
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Estrogen Response in Breast Cancer CAMA-1 Cells
dThd uptake set in when higher concentrations of estradiol
were used. At levels above 10 /¿Mestradiol, an inhibition of
dThd uptake to levels below that of control cells was often
encountered (data not shown).
5 •¿
A 24
H r Pioli
oÃ-ing
B 48 Hr Plat ing
5 -E
a. o
8x
Z S
CONTROL
8
INCUBATION
12
WITH
IS
ESTRADIOL
24
48
.2
I
,
( HR )
8
Chart 6. Time course of estrogen stimulation of dThd uptake into whole cells.
Cells were plated for 24 hr in 25% DCFBS prior to the addition of estradiol (E)
(10 HM), shown as 0 hr in the Chart. Following incubation with estradiol for the
period indicated, cells were rinsed with phosphate-buffered
saline twice, fresh
medium with 25% DCFBS was then added, and further incubation was com
menced to a total of 24 hr (from time of estradiol addition) for each flask. Thus,
all cells were incubated for a total of 48 hr. Control cells treated with (0) or
without O estradiol were incubated for a total of 48 hr without intermittent
washing. Two-hr dThd pulsing was conducted at the end of 48 hr (from time of
plating), according to the procedure in "Materials and Methods." Points, means
of triplicate determinations;
1982
16
20
24
I
(HR)
12
16
INCUBATION
C. 7 Days
2O
24
(HR)
Platine
90
•¿O
~
oars, S.D.
dThd, the effect of estrogen in stimulating a continuous uptake
of dThd into cells during a 7-day culture period was examined
(Chart 8). In the medium supplemented with 25% FBS, maximal
incorporation of dThd added during plating was reached by 48
hr; this level of dThd radioactivity was maintained in subsequent
days by CAMA-1 cells, possibly indicating that the rate of Sphase cell formation was balanced by the rate of cell death. In
cultures where cells were grown in media supplemented with
10 nw estradiol in 25% DCFBS, a much higher rate of dThd
uptake was observed during the first 48 hr. The subsequent
rate of cell death, however, was more rapid than the rate of
cell proliferation. Certain growth nutrients in FBS that were
removed by dextran-coated charcoal might have contributed
to the faster cell death. If estrogen effect were to increase the
pool size of dThd, this effect would be most noticeable during
the initial addition of dThd, and as equilibrium was reached,
this effect would be greatly reduced. From our results, estrogen
exerted a maximal rate of dThd uptake during the first 24 hr,
and a maximal level of uptake was reached at 48 hr following
the addition of dThd. It seemed unlikely that the late induction
by estrogen can be accounted for by the increase in pool size.
These results may best be explained by the ultimate increase
in the incorporation of dThd into DMA molecules, either through
DNA repair or through new synthesis. The finding from contin
uous labeling experiments is consistent with results of pulse
labeling (Charts 6 and 7), in which maximal rate of estrogen
stimulation also occurred within 24 hr after the addition of
estradiol.
The degree of induction of dThd uptake in DCFBS conditions
is dose dependent on estrogen. Almost a linear logarithmic
increase was observed from 10 PM to 10 nw (Chart 9). Although
sensitivity to estrogen varies with different passages of cells
and the amount of added DCFBS, this dose-dependent rela
tionship can be demonstrated repeatedly. The lowest level of
estradiol that is effective was usually at 10 pM. Following its
maximal induction, usually at 1.0 to 10 nM, depending on
incubation conditions and passages of cells, a reverse trend of
DECEMBER
12
INCUBATION
70
K - 60
O X
is».
8
12
16 20
24
INCUBATION (HR)
Chart 7. Sensitivity of 24-hr, 48-hr, and 7-day plated cells to estrogen stim
ulation. CAMA-1 cells of the same passage were plated for 24 hr (A). 48 hr (6).
and 7 days (C) in 25% DCFBS before the addition of 10 nw 17/i-estradiol (O) or
vehicle (•).Following each period of estrogen exposure, cells were pulsed with
dThd for 2 hr. Points, mean of triplicate determinations of radioactivity incorpo
rated per plate; oars. S.D.
to
Sao
o
20
er
O
o.
ce
o
o
10
_L
4
24
48
72
_1
_1
96
120 144
L
168
HOURS AFTER dThd ADDED
Chart 8. Effect of estradiol on continuous dThd uptake. CAMA-1 cells were
cultured either in 25% FBS CD or in 25% DCFBS + 10 nw estradiol (•).dThd
was added during plating (0 hr), and cells were harvested at each time point for
the determination of radioactivity. Each value is the mean of triplicate determi
nations; bars. S.D.
5063
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
8. S. Leung et al.
In the absence of serum, CAMA-1 cells attached poorly to
the surface of culture flasks, and cell death occurred precipi
tously 3 days following plating. A 10% serum substitute (12),
which contains constituents such as vitamins, fatty acids, phospholipids, amino acids, insulin, thyroxine, hydrocortisone, and
cholesterol, can maintain CAMA-1 cell growth. In fact, the rate
of cell proliferation in 10% serum substitute plus 1% DCFBS is
almost equivalent to that in 25% FBS during a 7-day culture
period (12). However, when dThd uptake was evaluated in a
E + PRL ( Sjig )
2 10
X
o.
o
OL
urd
E
2 t
C
12
10
E CONC. LOG (1/M
L
CONTROL
16-
5 2
)
Chart 9. Dose effect of estradici on precursor uptake. Different concentra
tions of estradici were added during plating. Following 48 hr in culture supple
mented with 25% DCFBS. cells were pulsed with 0.5 (iCi of dThd, leucine (LEU),
or undine (..mi for 2 hr. Controls were cells without the addition of estradiol.
Each value is the mean of triplicate determinations; bars, S.D. Each precursor
uptake experiment was conducted separately with different passages of cells.
18
o
J_
6
8
E ( 10 nM )
C
.12
10
8
6
E CONG. ( log 1/M )
Chart 11. Effect of estradici (E) in the absence of serum. Serum substitute
was prepared according to composition described previously (13). Estradici
and/or ovine prolactin (PRL) (P-S-12, a gift from National Institute of Arthritis,
Metabolism and Digestive Diseases) were added during plating m 15% serum
substitute to a final concentration as indicated in the chart. Following 48 hr in
culture, cells were pulsed with dThd as described in "Materials and Methods.
•¿
cultures with prolactin added; control cultures without any hormone added
O and with prolactin only (•)
were listed in C column.
I"
0.
Ü 12
O
l
9 -
10-
IT
O
O.
oc
o
u
o.
o
ce
D
8
16
24
HOURS E EXPOSURE
Chart 10. Effect of estradiol (£)[3H]uridine uptake following 8, 16, and 24 hr
of estradiol (10 nM) exposure. CAMA-1 cells were plated in 25% DCFBS for 24
hr prior to the addition of estradiol (10 nM). At the end of each period, cells were
pulsed with 5 »Ci| 'Hjuridme. Each column represents the mean of triplicate
Z 5
O
h<
4
O
o.
o:
O
O 3
determinations: bars, S.D.
Similar to dThd uptake, estrogen also stimulated the uptake
of [3H]uridine and [3H]leucine into cells as compared to controls
without added estradiol (Chart 9). The inductions again were
dose dependent. The time course of estrogen induction of [3H]
uridine is shown in Chart 10. No stimulation by estradiol was
observed at 8 hr, but significant stimulation was noted at 16and 24-hr exposures to estradiol added during plating.
5064
12
TIME AFTER
16
PLATING
20
24
(HR)
Chart 12. Effect of serum in stimulating dThd uptake in cultures supplemented
with 10% serum substitute and 1 nM estradiol. Serum (in percentages) was
added during plating (0 hr). At the end of each incubation time, cells were pulsed
•¿isual.
Points, mean of triplicate determinations; oars, S.D.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Estrogen Response in Breast Cancer CAMA-1 Cells
ERn
150
100
Ü
50
ERc
BSA
Õ
150
100
Q_
O
4S peak (Chart 13, (op). The ER peaks were abolished by an
excessive amount of diethylstilbestrol in the incubation mixture.
Besides stimulating precursor uptake and cell growth of
CAMA-1, estrogen also induced progesterone receptor biosyn
thesis (30). Tamoxifen and nafoxidine inhibited this process
(29).
DISCUSSION
50
10 15 20 25 Top
FRACTION
Chart 13. Sucrose gradient profiles of estrogen receptors of CAM A-1 cells.
At Day 7, cells from 8 T-75 flasks (5 X 106 cells/flask) were harvested by
scraping, homogenized with a glass-Teflon homogenizer in Tris-HCI-glycerolEDTA-dithiothreitol
buffer containing 20 mM sodium molybdate. Methods of
cytosol preparation and cytoplasmic ER [ERc determination (bottom) by sucrose
gradient technique were as described previously (15)], with the exception that
incubation was conducted for 4 hr at 0-4°; 2.5 nM [3H]estradiol was used for
total binding determinations (•);100-fold diethylstilbestrol was used as compet
itor for nonspecific binding determination (O); and sodium molybdate (20 mM)
was included in gradient buffers. Nuclear ER (ERn) (top) was extracted from
crude nuclear pellet with 04 M KCI Tris-HCI-glycerol-EDTA-dithiothreitol
buffer
for 1 hr at 4°with intermittent vortexing. KCI extract was used as the source for
nuclear ER determination according to cytoplasmic ER method described above.
"C-bovine serum albumin (BSA) was used as an internal marker.
straight serum substitute (Chart 11), estradiol could not elicit
a stimulatory effect. In many other experiments conducted on
dThd uptake, estradiol had no effect and only occasionally
showed a marginal stimulation when estrogen-treated
cells
were compared to control cells. Nevertheless, high levels of
estradiol always were inhibitory. The level of estradiol that was
necessary to achieve inhibition was usually about 10-fold lower
than that required under DCFBS conditions. Interestingly, the
addition of prolactin reestablished, at least partially, the stim
DECEMBER
ulatory effect and reduced the inhibitory effect of estradiol. In
this regard, it may be speculated that prolactin might be in
volved in the enhancement of cell cycle transverse elicited by
estradiol.
The effect of serum in a nutritionally adequate environment
O.e., 10% serum substitute) was studied, and the time course
of dThd uptake due to different proportions of DCFBS in 10%
serum substitute and 1 nw estradiol was determined (Chart
12). With 1% DCFBS added, the rate of dThd uptake was low
and changed only slightly from 8 to 12 hr following plating. A
50% increase in dThd uptake was observed during the same
period in a 5% DCFBS condition. Further increase in dThd
uptake was noted with increased proportions of added DCFBS.
Results from this experiment indicate that factors promoting a
more rapid rate of cell cycle traverse, for example from Gìto
S phase, are present in the serum. The relationship of estradiol
to these factors, however, requires further experimentation.
It is known that estrogen target tissues contain ER. CAMA-1
cells contain cytoplasmic ER which sediment at the 8S region
of a sucrose gradient (Chart 13, bottom). Most of the ER
complexes were present in the crude nuclear fraction and
could be extracted by a high-ionic-strength
buffer, yielding a
1982
Since the successful culture of an estrogen-responsive cell
line (MCF-7) in long-term culture (25), many studies have been
conducted to investigate the role of hormones in this cell line
(3, 5, 9, 10, 16-18, 21, 28). Other established human breast
cancer cell lines that are estrogen sensitive have also been
reported (1, 9). These cells, similar to other estrogen target
tissues, contain cytoplasmic or nuclear ERs (7, 9, 16). In this
report, we present findings of another established cell line of
human origin that responds to estrogen in culture. Results
presented in this communication show that CAMA-1 cells de
pend on estrogen for growth and have the characteristics of an
estrogen-dependent cell line. When CAMA-1 cells are grown
in DCFBS, cell proliferation is markedly reduced. Chronic in
sufficiency of estrogen added to cultures also leads to an
apparent loss of response of CAMA-1 cells to estrogen-induced
dThd uptake (30).
Several investigations have pointed out that estrogen-in
duced cell proliferation may be mediated through estrogeninduced proteins (20, 23, 24). When CAMA-1 cultures are not
supplemented with serum, estrogen cannot elicit a stimulatory
effect on dThd uptake. From data presented herein and else
where (12), the lack of estrogenic stimulation of dThd uptake
in serum substitute appears to be from factors other than
nutritional, since CAMA-1 cells proliferate as well in serum
substitute as in serum (12). Our results demonstrate that serum
factors or proteins may be necessary for estrogen-induced
growth in vitro. It remains to be tested whether these sub
stances are related to the estrogen-induced proteins (23, 24)
that were described to mediate estrogen action in vivo.
The bipnasic response of CAMA-1 cells to estrogen-induced
5065
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
ß.S. Leung et al.
dThd uptake has been reported by others for MCF-7 cell line
(16). It is tempting to relate this characteristic response of cells
in vitro to clinical trials, where low levels of estrogen exacerbate
breast cancer in patients, while high levels often provide ben
eficial palliation. However, high levels of estradici in vitro may
simply exert a nonspecific cytotoxic effect upon culture cells.
Further experimentation is needed to demonstrate whether or
not this inhibitory effect is specific to estrogen-sensitive cells.
ERs in CAMA-1 cells are largely located in the nucleus.
These results are consistent with findings reported for other
cell lines (7, 9). This phenomenon, however, is different from
normal estrogen target tissues such as the uterus, where more
than 70% of receptors are present in the cytoplasm. Similarly,
human breast cancers also contain high levels of cytoplasmic
receptors (11,19). These results seem to indicate that culture
conditions or serum factors might have altered the distribution
of ER. It was suggested (8) that the level of nuclear receptors
in cultured cells is related to estrogen induction of progester
one receptors. Although CAMA-1 cells also exhibit a dose
response to estrogen induction of progesterone receptor (30),
the quantitative relationship of nuclear receptor and estrogeninduced progesterone receptor and other biological responses
requires further experimentation.
We have demonstrated that estrogen not only stimulates
proliferation of CAMA-1 cells, it also stimulates cellular events
such as uptake of dThd, [3H]leucine, and [3H]uridine, and the
induction of progesterone receptors (30). In other studies, we
have shown that the incorporation of oleoyl-CoA into phosphatidylinositols and phosphatidylcholines
are also dependent
upon estrogen concentration (26). In addition to estrogen,
CAMA-1 cells respond to other hormones such as prolactin,
progesterone, insulin (12), glucocorticoid, and androgen (6) in
culture. Estrogen-induced cellular events such as protein syn
thesis (28) and progesterone receptor production (8, 9) have
also been reported for the MCF-7 cell line. Specific estrogeninduced proteins in CAMA-1 cells are being actively investi
gated in our laboratory.
In conclusion, CAMA-1 cell line has the unique characteris
tics of an estrogen-dependent
tissue. It may provide a good
alternative model for the elucidation of estrogen action in cell
proliferation of human breast cancer. The feasibility of using
this cell line to understand the interaction of prolactin, estrogen,
and progesterone in breast cancer is encouraging (12), and
investigations related to the interaction of these hormones in
CAMA-1 cells are in progress. The effects of these hormones
on CAMA-1 cells in nude mice remain to be explored.
ACKNOWLEDGMENTS
We thank Dr. J. Fogh of Sloan-Kettering Institute for Cancer Research for the
generous gift of CAMA-1 cell line, the National Institute of Arthritis. Metabolism
and Digestive Diseases for the gift of ovine prolactin. and Anne Potter for
excellent technical assistance.
REFERENCES
1. Allegra. J. C., and Lippman. M. E. Growth of a human breast cancer cell line
in serum-free hormone-supplemented
medium. Cancer Res.. 38. 38233829, 1978.
2. Bradley, C. J.. Kledzik, G. S., and Meites. J. Prolactin and estrogen de
pendency of rat mammary cancers at early and late stages of development.
Cancer Res., 36. 319-324, 1976.
5066
3. Burke. R. E., and McGuire, W. L. Nuclear thyroid hormone receptors in a
human breast cancer cell line. Cancer Res.. 38. 3769-3773,
1978.
4. Fogh, J., Wright, W. C., and Loveless, J. D. Absence of HeLa cell contami
nation in 169 cell lines derived from human tumors. J. Nati. Cancer Inst,
58. 209-214, 1977.
5. Gaffney. E. V.. Pigott, D. A., and Grimaldi, M. A. Human serum and growth
of human mammary cells. J. Nati. Cancer Inst., 63. 913-918, 1979.
6. Gao, Y. L., Leung, B. S.. Potter, A. H., and Yu, W. C. Y. Effects of steroid
and peptide hormones on human mammary carcinoma cell proliferation in
long-term culture. J. Minn. Acad. Sci., in press, 1982.
7. Geier, A., Cocos, M.. Ginzburg, R., Haimsohn, M., and Lunenfeld, B.
Estradici binding to nuclear receptors in human breast cancer tissue (MCF7 cell line) and in dimethylbenz(a)anthracene-induced
mammary carcinoma.
J. Clin. Endocrinol. Metab., 49: 34-39. 1979.
8. Horwitz. K. B., and McGuire, W. L. Estrogen control of progesterone recep
tors in human breast cancer. Correlation with nuclear processing of estrogen
receptor. J. Biol. Chem., 253. 2223-2228.
1978.
9. Horwitz, K. B., Zava, D. T., Thilagar, A. K., Jensen, E. M.. and McGuire, W.
L. Steroid receptor analyses of nine human breast cancer cell lines. Cancer
Res., 38. 2434-2437,
1978.
10. Jozan, S., Moure, C., Gillois, M., and Bayard. F. Effects of estrone on cell
proliferation of human breast cancer (MCF-7) in long term tissue culture. J.
Steroid Biochem., rO: 341-342, 1979.
11. Leung, B. S. Hormonal dependency of experimental breast cancer. In: W. L.
McGuire (ed.), Hormones, Receptors, and Breast Cancer, pp. 219-261.
New York: Raven Press, 1978.
12. Leung, B. S. Interaction of prolactin. estrogen and progesterone in a human
mammary carcinoma cell line. CAMA-1.1. Cell growth and thymidine uptake.
J. Steroid Biochem., /5. 421-427, 1981.
13. Leung, B. S. (ed.). Hormonal Regulation of Experimental Mammary Tumor.
Vols. 1 and 2. Quebec, Quebec. Canada: Eden Press, in press, 1981.
14. Leung, B. S., Qureshi, S., and Leung, J. S. Effects of prolactin and estrogen
on human breast cancer cells in long-term tissue culture. Abstract 112.
Sixtieth Annual Meeting of the Endocrine Society. 1978.
15. Leung, B. S., and Sasaki, G. H. On the mechanisms of prolactin and estrogen
action in 7,12 dimethylbenz(a)anthracene-induced
mammary carcinoma in
the rat. II. In vivo tumor responses and estrogen receptor. Endocrinology,
97. 564-572, 1975.
16. Lippman, M., Bolán, G., and Huff, K. The effects of estrogens and antiestrogens on hormone-responsive
human breast cancer in long-term tissue
culture. Cancer Res., 36. 4595-4601.
1976.
17. Lippman, M., Bolán. G., Monaco, M., Pinkus, L., and Engel. L. Model
systems for the study of estrogen action in tissue culture. J. Steroid Bio
chem., 7: 1045-1051,
1976.
18. Lippman, M., Monaco, M. E., and Bolán, G. Effects of estrone, estradiol,
and estriol on hormone-responsive human breast cancer in long-term tissue
culture. Cancer Res., 37. 1901-1907,
1977.
19. McGuire, W. L.. Chamness, G. C., Costlow, M. E., and Shepherd. R. E.
Hormone dependence in breast cancer. Metab. Clin. Exp.. 23. 75-100,
1974.
20. Nair, B. K., and DeOme, K. B. A growth-stimulating factor from solid mouse
mammary tumors. Cancer Res., 33. 3222-3226,
1973.
21. Shafie, S., and Brooks, S. C. Effect of prolactin on growth and the estrogen
receptor level of human breast cancer cells (MCF-7). Cancer Res., 37. 792799, 1977.
22. Sinha. D., Cooper. D., and Dao. T. L. The nature of estrogen and prolactin
effect on mammary tumorigenesis. Cancer Res., 33. 411-414, 1973.
23. Sirbasku, D. A. Estrogen induction of growth factors specific for hormoneresponsive mammary, pituitary, and kidney tumor cells. Proc. Nati. Acad.
Sei. U. S. A., 75: 3786-3790,
1978.
24. Sonnenschein, D..and Soto, A. M. But are estrogen per se growth-promoting
hormones? J. Nati. Cancer Inst., 64: 211-215, 1980.
25. Soule, H. D., Vazquez. J., Long, A.. Albert, S., and Brennan. M. A human
cell line from a pleural effusion derived from a breast carcinoma. J. Nati.
Cancer Inst., 51: 1409-1413,
1973.
26. Sun, G. Y., and Leung, B. S. Prostaglandin, fatty acids and phospholipids in
normal and neoplastic breast tissues. In: B. S. Leung (ed.). Hormonal
Regulation of Experimental Mammary Tumors. Vol. 2. Quebec, Quebec,
Canada: Eden Press, in press. 1981.
27. Welsch, C. W.. and Nagasawa, H. Prolactin and murine mammary tumori
genesis: a review. Cancer Res., 37 951-963, 1977.
28. Westley. B., and Rochefort. H. Estradiol induced proteins in the MCF-7
human breast cancer cell line. Biochem. Biophys. Res. Commun., 90: 410416, 1979.
29. Yu, W., and Leung, B. S. Estrogen and antiestrogen effects in progesterone
receptor levels in human mammary carcinoma cells, CAMA-1. Fed. Proc..
39: 2134, 1980.
30. Yu, W. C. Y., Leung, B. S., and Gao, Y. L. Effects of 17/î-estradiol on
progesterone receptors and the uptake of thymidine in human cancer cell
line CAMA-1. Cancer Res., 41: 5004-5009,
1981.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Response to Estrogen by the Human Mammary Carcinoma Cell
Line CAMA-1
Benjamin S. Leung, Shehla Qureshi and Jonas S. Leung
Cancer Res 1982;42:5060-5066.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/12/5060
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1982 American Association for Cancer Research.