[CANCER RESEARCH 38, 3823-3829,
0008-5472/78/0038-0000$02.00
November 1978]
Growth of a Human Breast Cancer Cell Line in Serum-free Hormonesupplemented Medium
Joseph C. Allegra and Marc E. Lippman1
Medicine Branch, National Cancer Institute. National Institutes of Health, Bethesda, Maryland 20014
MATERIALS
ABSTRACT
AND METHODS
Cells. The ZR-75-1 cell line (6) was used for all studies. It
ZR-75-1, a human breast cancer cell line, has been
grown in hormone-supplemented medium without serum. is a human breast cancer cell line derived from a malignant
The factors required for optimal growth include 17/3-estra- ascitic effusion of a patient with infiltrating duct carcinoma
diol, insulin, transferrin, dexamethasone, and L-triiodo- of the breast. The cell line has been grown in MEM2 (3)
thyronine. If estradiol, insulin, or L-triiodothyronine is supplemented with 5 to 10% fetal calf serum for more than
omitted, cells cease division within 7 days, but viability is 2.5 years and through 68 passages. Its morphological and
retained for at least 14 days. Omission of transferrin leads karyotypic characteristics are similar to the original breast
to cell death within 7 days. The cells have been continu tumor specimen.
Medium and Hormones. IMEM (18) supplemented with Lously maintained in this environment without morphologi
cal alteration or cessation of growth for more than 5 glutamine (0.6 g/liter), penicillin (62 mg/liter), and strep
months. Addition of the anti-estrogen, Tamoxifen (10 6 tomycin (135 mg/liter) was the basic culture medium to
M), inhibited cells below the growth rate seen when which hormones and growth factors were added. All media
were prepared in the National Institutes of Health Media
estradiol was omitted from the medium, even when Ta
moxifen was added 4 days and two medium changes after Unit. Transferrin (Sigma Chemical Co., St. Louis, Mo.) was
the removal of estradiol from the medium, thus suggesting added at a final concentration of 1 ¿ig/ml(10"" M). L-Trian action of Tamoxifen which may be independent of iodothyronine (Sigma) (10~5M) stock solution was prepared
competition with estradiol. The availability of a human in 0.1 N NaOH and added to medium to yield a final con
breast cancer cell line that can be propagated in hormone- centration of 10~8M. Insulin U-100 (Eli Lilly & Co., Indianap
supplemented medium without serum should aid in the olis, Ind.) was added at a concentration of 5 x 10~7M. 17/3study of the mechanisms by which hormones effect cell Estradiol and dexamethasone (Sigma) in benzene-ethanol
growth.
were evaporated to dryness, dissolved in ethanol, and
stored at -20° until use. Final concentrations in the me
dium were 10~8 M 17/3-estradiol and 10~8 M dexametha
INTRODUCTION
It has recently been shown that it is possible to grow
some cell lines in medium without added serum, provided
this culture medium is supplemented with hormones and
other factors (9, 11). For example, Hayashi ef al. (10) have
shown that the GH3rat pituitary cell line will grow in Ham's
Nutrient Mixture F-12 supplemented with L-triiodothyro
nine, thyrotropin-releasing hormone, transferrin, parathy
roid hormone, insulin, fibroblast growth factor, and somatomedin C. Even after long-term culture, the growth in this
hormone-supplemented medium is equivalent to growth in
serum-supplemented medium. Several other cell lines have
been adapted to serum-free growth (12, 14, 19).
We felt that the availability of a human breast cancer cell
line that could be propagated in serum-free defined medium
would aid in the study of the mechanisms of hormone
interaction with breast cancer cells. The ZR-75-1 cell line
was chosen because we have previously demonstrated
receptors for and responses to estrogen, insulin, androgen,
and glucocorticoid and because of the unequivocal bio
chemical, morphological, and chromosomal evidence of its
human breast cancer origin (6, 16). We were particularly
anxious to develop such a defined growth system in order
to be able to study specific hormonal effects.
1 To whom requests for reprints should be addressed, at Building 10,
Room 6B02, Medicine Branch, National Cancer Institute, National Institutes
of Health, Bethesda, Maryland 20014.
NOVEMBER
sone. The final concentration of ethanol is 0.1%, and this
concentration has no effect on the growth of the cells.
Tamoxifen (ICI 41474) was similarly prepared. Fibroblast
growth factor (Collaborative Research, Waltham, Mass.) at
a concentration of 0.025 /¿g/mlwas added to the tissue
culture flasks when the cells were subcultured, in addition
to nucleosides [10~8 M cytidine, uridine, thymidine, and
adenosine (Sigma)] and nonessential amino acids (L-alanine, 18 mg/liter; L-asparagine-H2O, 30 mg/liter; u-aspartic
acid, 27 mg/liter; L-glutamic acid, 29 mg/liter; glycine, 15
mg/liter; L-proline, 23 mg/liter; L-serine, 21 mg/liter). Other
factors tested for growth-promoting activity include epider
mal growth factor (Collaborative Research), 5a-d ihydro tes
tosterone (Steraloids Inc., Pawling, N. J.), vasopressin
(Calbiochem, Gaithersburg, Md.), oxytocin (Sigma), and
human placental lactogen (Sigma).
Cell Growth Experiments. Cells growing exponentially in
MEM + 5% fetal calf serum were suspended with trypsinEDTA (trypsin, 0.05%; EDTA, 0.02%) and were plated in
replicate in MEM supplemented with 5% charcoal-treated
calf serum (2). The cells were plated into sterile 6-well (35mm) plastic tissue culture dishes (Linbro Scientific Inc.,
Hamden, Conn.). After sufficient time for the cells to be
come adherent had elapsed (usually 12 to 16 hr), the
' The abbreviations used are: MEM, minimal essential medium; IMEM,
improved minimal essential medium; IMEM-HS, improved minimal essential
medium, hormone supplemented.
1978
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3823
J. C. Allegra and M. E. Lippman
medium was changed to improved minimal essential me
dium supplemented with estradiol, L-triiodothyronine, in
sulin, dexamethasone, and transferrin (IMEM-HS), or to
IMEM-HS minus a specific hormone. After 24 hr, the me
dium was replenished with fresh medium of identical com
position. At various times, cells were collected by suspen
sion in trypsin-EDTA and counted in a hemocytometer.
Cell Transfer. The serum-free cells are transferred by
treating with 0.02% EDTA in saline A (NaHC03, 0.35 g/liter;
NaCI, 8.0 g/liter; KCI, 0.4 g/liter; dextrose, 1.0 g/liter; and
phenol red, 0.02 g/liter). The cell layer was rinsed with this
solution and the excess removed. When the cells began to
detach, the action of the EDTA was stopped by simple
dilution in IMEM-HS. Cells were passed at a 1:2 dilution.
Plating efficiency experiments were performed by plating
25,000 cells in a 75-sq cm tissue culture flask. After 7 days,
the cell colonies were counted. Plating efficiency is defined
as the number of colonies formed x 100 divided by the
number of cells added to the tissue culture flask.
Precursor Incorporation. To assessthe effect of Tamoxifen on thymidine incorporation in the ZR-75-1 breast cancer
cells growing in IMEM-HS minus 17/3-estradiol, radiolabeled thymidine (Amersham-Searle, Arlington Heights, III.)
diluted in Dulbecco's phosphate-buffered saline (KCI, 0.20
g/liter; KH2PO4,0.20 g/liter; NaCI, 8.0 g/liter; Na2HPO4-7
H,O, 2.16 g/liter, pH 7.4) was added to each dish 1 to 2 hr
before the cells were harvested. Each dish usually con
tained 1 /¿Ci
of tritium. Cells were harvested by washing the
dishes once with Dulbecco's phosphate-buffered saline,
suspending in EDTA (0.02%), and collecting cell pellets by
centrifugation. Cell pellets were suspended in water and
sonically dispersed for 3 sec in a Branson sonic extractor
(Branson Sonic Power Co., Danbury, Conn.) at the lowest
setting. Aliquots were then used for the determination of
protein by the method of Lowry (15) or thymidine incorpora
tion by precipitation in 10% trichloroacetic acid. Acid-in
soluble counts were collected and washed on a 0.45-/u.m
Millipore filter. After drying, the filters were solubilized
in Aquasol (New England Nuclear, Boston, Mass.) and
counted in a Packard scintillation counter (Packard Instru
ment Co., Downers Grove, III.) (efficiency for tritium,
-35%).
crease the growth of cells already growing rapidly in IMEMHS. Chart 2 compares the growth of the cells in IMEM-HS
and IMEM-HS supplemented with 5% fetal calf serum.
Again, over this 9-day period no differences in growth were
observed, and thus we conclude that the addition of fetal
calf serum to IMEM-HS does not result in more rapid growth
of these breast cancer cells.
The effects of each of the hormones and transferrin on
growth are assessed in Chart 3. As can be seen in this
chart, the cells in IMEM-HS grow rapidly over the 14-day
period. Control cells in IMEM alone remain viable for 4 to 7
days as judged by attachment to the plastic tissue culture
dishes, and then detach and die, as do the cells in IMEMHS that lack transferrin. Cells in IMEM-HS minus estradici
or insulin or L-triiodothyronine appear to grow for approxi
mately 7 days and then become static, although viability is
maintained for the 14-day period. The addition or subtrac
tion of 10~" M dexamethasone had no apparent affect on
growth but appeared to increase plating efficiency and thus
was included in IMEM-HS (see below).
The dose-response curve for transferrin is illustrated in
Chart 4. As low a concentration as 0.25 jug/ml (2.5 x 10~12
•¿
IMEM-HS
O IMEM PLUS 5% FETALCALF SERUM
£ 60
OAY
Chart 1. Growth of the ZR-75-1 cell line in hormone-supplemented me
dium without serum. Cells were plated at a density of 50,000 cells/dish in
MEM supplemented with 5% charcoal-treated calf serum. On Day 1, medium
was changed to IMEM-HS (•)
and IMEM + 5% fetal calf serum (D). Arrows,
days on which the cells were refed with fresh medium. Standard deviations
of triplicate cell counts shown are generally less than 10%.
•¿
IMEM-HS
O IMEM-HS PLUS 5% FETALCALF SERUM
RESULTS
Growth Experiments. ZR-75-1 cells grow rapidly in IMEMHS. In Chart 1, the growth of the ZR-75-1 cells in IMEM-HS
is compared to their growth in IMEM supplemented with an
optimal concentration of fetal calf serum (5%). In this
experiment the cells in IMEM-HS are growing at a more
rapid rate than those in fetal calf serum. In other experi
ments not shown, their growth rates are usually equal. As
will be shown later, if hormones and serum are omitted, the
cells die. The ZR-75-1 human breast cancer cell line grows
rapidly in improved MEM supplemented with hormones and
the iron transport protein transferrin. The optimal concen
tration of 17/3-estradiol in IMEM-HS has previously been
shown to be 10~8 M (6). As described later, 5 x 10~7 M
insulin, 10~" M L-triiodothyronine, 10~8 M dexamethasone,
and 10" M transferrin (1 ¿ig/ml) are optimal for cell
growth.
We next wondered whether fetal calf serum could in3824
« 8
Å’
£
¡
6
Chart 2. Growth of the ZR-75-1 cell line in hormone-supplemented me
dium without serum. Cells were plated at a density of 1000 cells/dish in
MEM-supplemented with 5% charcoal-treated calf serum. On Day 1, the
medium was changed to IMEM-HS (•)
and IMEM-HS supplemented with 5%
fetal calf serum (O). Arrows, days on which the cells were refed with fresh
medium. Standard deviations of triplicate cell counts are generally less than
10%.
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Cell Growth in Hormone-supplemented
M) causes an increase in cell number after 4 days, compared
to control cells. The curve has a slow Increase and is
relatively flat between the concentration range of 1 to 7 ¿ig/
ml (1Q-11to 7 x IO"11 M). Although this experiment demon
strates that addition of transferrin leads to an increase in
cell number, the absolute dependence of the cells for
transferrin is better demonstrated in the growth curve
shown in Chart 3.
30 r
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o
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co5D
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\H,-'X/
70
-^
•¿//lililÃ-
Z—
iOl
—¿
+
Medium
V
—¿IMEM-HS
10Jf
•¿
60
o
SO
IMEM-HSMINUS17/ÃŽESTRADIOL
—¿-*--
IMEM-HSMINUSINSULIN
—¿O~
IMEM-HSMINUST,
—¿-^-—
IMEM-HS
—¿O—
IMEM
MINUSTRANSFERRIN
10-'°
io-*
IO"1
io-1 io-*
T, CONCENTRATION IMI
Chart 5. Effect of L-triiodothyronine on growth of ZR-75-1 cells. Cells
were plated at a density of 50,000 cells/dish in MEM supplemented with 5%
charcoal-treated calf serum. After sufficient time for the cells to become
adherent had elapsed, the medium was changed to IMEM-HS with varying
concentrations of L-triiodothyronine. After 24 hr, the medium was exchanged
for fresh medium, and the experiment was allowed to continue for 4 days, at
which time the cells were harvested and counted. Standard deviations of
triplicate cell counts are generally less than 10%.
f
2
I
!
10
t
12
14
DAY
Chart 3. Requirements for serum-free growth of ZR-75-1 human breast
cancer cells. Cells were plated at a density of 50,000 cells/dish in MEM
supplemented with 5% charcoal-treated calf serum. Over the next 4 days,
the medium was exchanged twice with fresh MEM plus 5% charcoal-treated
calf serum. On Day 0, the medium was changed to IMEM-HS, IMEM-HS
minus 17/3-estradiol, IMEM-HS minus insulin, IMEM-HS minus T3, IMEM-HS
minus transferrin, or IMEM. Arrows, days on which the cells were refed with
fresh medium. Standard deviations of triplicate cell counts are generally less
than 10%.
r
o
2
Chart 5 shows the dose-response curve for L-triiodothy
ronine in IMEM supplemented with all of the other required
growth factors. As can be seen in this chart, an effect on
growth of L-triiodothyronine is observed at concentrations
as low as 10~10 M with maximal stimulatory effects on
growth at 10~8 M. The effect on growth is diminished at
higher concentrations, and 1LV6 and 10 5 M exhibit no
growth-promoting activity.
The dose-response curve for insulin is shown in Chart 6.
Insulin in varying concentrations was added to IMEM sup
plemented with all of the other required growth factors. An
effect by insulin on growth is observed at concentrations as
low as 10~11M. The growth-promoting effect is maximal at
IO'10 M, and this maximal effect persists at a concentration
range between 10 10and 10~6M.
The effect of other factors on growth of the ZR-75-1 cells
was also investigated. No further growth-promoting activity
was observed when the nucleosides, adenosine (10~8 M),
uridine (10~8 M), thymidine (10~8 M), or cytidine (10 * M),
20
i
O '
io-io
TRANSFERRINCONCENTRATIONIMI
Chart 4. Effect of transferrin on growth of ZR-75-1 cells. Cells were plated
at a density of 50,000 cells/dish in MEM supplemented with 5% charcoaltreated calf serum. After sufficient time for the cells to become adherent had
elapsed, the medium was changed to IMEM-HS with varying concentrations
of transferrin. After 24 hr, the medium was exchanged for identical fresh
medium, and the experiment was allowed to continue for 4 days, at which
time the cells were harvested and counted. Standard deviations of triplicate
cell counts are generally less than 10%.
NOVEMBER
nonessential amino acids, human placental lactogen (2 ¿ig/
ml), fibroblast growth factor (0.025 ¿ig/ml), epidermal
growth factor (0.01 pig/ml), vasopressin (1 milliunit/ml),
oxytocin (1 milliunit/ml), or5a-dihydrotestosterone (10"B M)
was added to IMEM-HS.
The effect of the antiestrogen, Tamoxifen, on thymidine
incorporation and growth of the ZR-75-1 cells is examined
in Chart 7. In this experiment, the cells were plated in MEM
plus 5% charcoal calf serum. Charcoal-treated calf serum
was used because it is steroid depleted and contains very
low concentrations of estrogen. This medium was ex
changed twice over a 4-day period, and the medium was
then changed to either IMEM-HS minus 17/3-estradiol or
IMEM-HS minus 17/3-estradiol plus 10 6 M Tamoxifen. Over
the 10-day period there was a difference in cell number,
suggesting that Tamoxifen has an action on the breast
1978
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3825
J. C. Allegra
and M. E. Lippman
Cell Transfer and Long-Term Culture. The transfer of the
ZR-75-1 cell line in IMEM-HS is difficult. Cells are passed at
a dilution ratio of 1:2 because of a low plating efficiency.
Table 1 lists the results of the plating efficiency experi
ments. The plating efficiency of the cells in IMEM-HS is
approximately 1%. This can be improved to approximately
3 to 4% by the addition of nonessential amino acids,
nucleosides (10~8 M adenosine, cytodine, thymidine, uri-
14
12
10
Co
5
-12
10
IO'1
.-IO
io-'
io-
INSULIN CONCENTRATION (Ml
Chart 6. Effect of insulin on growth of ZR-75-1 cells. Cells were plated at
a density of 200,000 cells/dish in MEM supplemented with 5% charcoaltreated calf serum. After sufficient time for the cells to become adherent had
elapsed, the medium was changed to IMEM-HS with varying concentrations
of insulin. After 24 hr, the medium was exchanged for fresh identical
medium, and the experiment was allowed to continue for 3 days, at which
time the cells were harvested and counted. Standard deviations of triplicate
cell counts are generally less than 10%.
t
t
t
dine), and fibroblast growth factor, 0.025 /¿g/ml.We rou
tinely add these factors at the time of subculture and, since
none of these factors appear to have any growth-promoting
activity on the ZR-75-1 cells, the flasks are refed with fresh
IMEM-HS after plating has occurred. The plating efficiency
of the cells in IMEM alone is less than 0.1% and is not
significantly improved by the addition of bovine serum
albumin (100 /xg/ml). The plating efficiency of the ZR-75-1
cells in IMEM plus 5% fetal calf serum is greater than 50%.
If one omits dexamethasone from IMEM-HS, the plating
efficiency decreases to 0.1%.
We also attempted to improve plating efficiency by the
addition of conditioned medium. We have preliminary data
that the ZR-75-1 cells condition their medium and that this
conditioned medium, when added to the cells, leads to an
increase in thymidine incorporation at 24 hr and an increase
in cell number at 48 hr (Allegra and Lippman, unpublished
data). As can be seen in the table, 50% (by volume) condi
tioned medium did not increase plating efficiency when
added to IMEM-HS or IMEM-HS supplemented with nucleo
sides, nonessential amino acids, or fibroblast growth fac
tor.
Thus far, the cells have been carried in IMEM-HS for 5
months and through 13 passages. No morphological
changes have been observed during this period, and there
has been no alteration in growth rate. Figures 1 and 2
compare control cells that have been growing continuously
in MEM + 5% fetal calf serum with cells that have been
growing in IMEM-HS for 3 months. No significant differ
ences can be seen.
DISCUSSION
Growth of cells in a defined medium without serum
Chart 7. The effect of Tamoxifen on ZR-75-1 human breast cancer cells.
Cells were plated at density of = 50.000 cells/dish in MEM + 5% charcoalsupplementation has been a goal in tissue culture for many
treated calf serum. Over the next 4 days, the medium was exchanged twice
with fresh MEM + 5% charcoal-treated calf serum. On Day 0, the medium
was changed to IMEM-HS minus 170-estradiol or IMEM-HS minus 170Table 1
estradiol plus 10 •¿
M Tamoxifen. Arrows, days on which the cells were fed.
Effect of factors on plating efficiency of ZR-75-1 human breast
Standard deviations of triplicate cell counts and thymidine incorporation are
cancer cells
generally less than 10%. •¿
IMEM-HS minus 17/3-estradiol (cell number); O,
IMEM-HS minus 17/3-estradiol plus IO " M Tamoxifen (cell number); •¿
Plating efficiency
IMEM-HS minus 17/3-estradiol (thymidine incorporation); D, IMEM-HS
Medium
(%)
minus 170-estradiol plus 10 •¿
M Tamoxifen (thymidine incorporation).
cancer cells which may be totally independent of competi
tion with estradiol. Despite prolonged incubation in estradiol-free medium, it is not possible to exclude the possibility
that some (or all) of the Tamoxifen effect may still reflect
interference with the action of occultly retained estradiol.
Note that at Day 10 there is a 2-fold decrease in cell number
of the ZR-75-1 cells in medium containing Tamoxifen and a
>50% decrease in thymidine incorporation. It is interesting
to note that thymidine incorporation decreases as cell
density increases.
3826
IMEM
IMEM plus bovine serum albumin
IMEM-HS
IMEM-HS minus dexamethasone
IMEM-HS plus bovine serum albumin
IMEM-HS plus nucleosides
IMEM-HS plus nonessential amino acids
IMEM-HS plus fibroblast growth factor
IMEM-HS plus nucleosides, nonessential
amino acids and fibroblast growth factor
IMEM-HS plus nucleosides, nonessential
amino acids, fibroblast growth factor, and
conditioned medium
01
0.1
1.2
0.1
0.8
1.1
1.0
1.8
3.4
3.2
CANCER RESEARCH VOL. 38
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Cell Growth in Hormone-supplemented
years. The requirement of virtually all established cell lines
for serum has hampered the investigation of many aspects
of hormone action. Cells growing in defined medium would
be of major importance since they represent an ideal system
for the study of cell biology in the absence of undefined
serum factors.
The first major contribution in this area was the definition
by Eagle (5) of the basic nutritional requirements of cells in
tissue culture. This was followed by the formulation of
many different synthetic media which aided in cell growth
(8, 21). Since the advent of these improved synthetic media,
a great deal of effort has been expended in trying to find
optimal culture conditions for cell growth in totally defined
serum-free medium (1, 7,13). Higuchi (12) was able to grow
animal cells in chemically defined media, and Katsuta and
Takaoka (14) were also successful in the cultivation of
animal cells in a synthetic medium that was free of both
protein and lipid. Both Higuchi (12) and Katsuta and Tak
aoka (14) review other cell lines which have been adapted
to defined medium conditions.
In an attempt to study differentiated cell functions,
Thompson et al. (19) were able to successfully grow the rat
hepatoma cell line, HTC (20), in a chemically defined
medium without macromolecular supplementation. Follow
ing a short period of adaption (1 to 2 weeks), the HTC cells
grew exponentially in IMEM-zinc option (18), and both their
appearance and rate of growth were similar to that of their
serum-grown counterparts. Using this system, these au
thors were able to define the amino acid requirements for
growth of the HTC cells and also were able to study the
regulation of hepatic tyrosine aminotransferase and its
induction by glucocorticoids and insulin under serum-free
conditions. They demonstrated that serum was not required
for hepatic tyrosine aminotransferase induction by insulin
or glucocorticoids. Recently, Burks and Peck (4) reported
on a serum-free medium that supports the proliferation of
isolated bone cells in primary culture. In this defined sys
tem they were able to study the differentiated cell function
of alkaline phosphatase activity and also cellular response
to parathyroid hormone as measured by increased levels of
cyclic AMP.
Hayashi ef al. (10) have shown that it is possible to omit
serum from culture medium and to sustain cellular growth,
provided the culture medium is supplemented with hor
mones and other growth-promoting factors. These authors
feel that the major role of serum supplementation of culture
medium is to provide hormones necessary for growth. The
GK, rat pituitary cell line can be grown in serum-free
medium (Ham's Nutrient Mixture F-12) (8) supplemented
with L-triiodothyronine,
thyrotropin-releasing
hormone,
transferrin, parathyroid hormone, insulin, fibroblast growth
factor, and somatomedin C. Even after long-term culture,
the growth in this hormone-supplemented medium is equiv
alent to growth in serum-supplemented medium. These
same authors have also successfully grown the HeLa cell
line and the M2R mouse melanoma cell line in hormonesupplemented defined medium. In comparing the hormone
requirements for the three cell lines, it was noted that thus
far all have required insulin and transferrin, although their
effect on growth at identical concentrations varies among
the cell lines and each cell line has a requirement for a
NOVEMBER
Medium
hormone which localizes in the nucleus, such as L-triiodo
thyronine or a steroid. However, the HTC cell line which
grows without adaption in defined medium neither requires
hormone supplementation of any kind nor does it "condi
tion" the medium (19). The presence of an unstable condi
tioning component cannot be completely excluded in that,
in defined medium, the HTC cells have a lower plating
efficiency.
The adaption of the ZR-75-1 cell line to serum-free growth
has many potential benefits. It is a human breast cancer cell
line and it has receptors for estrogen, androgen, insulin,
glucocorticoid, and progesterone and responds to estro
gen, androgen, insulin, and glucocorticoid (6, 16). We felt
that the establishment of the cell line in defined medium
without the unknown effects of serum factors would better
enable investigators to study the mechanisms of hormone
interaction with breast cancer. Our preliminary data on the
effect of the antiestrogen, Tamoxifen, are encouraging. In
an environment in which extensive incubation in media
devoid of estrogen (IMEM-HS minus 17ß-estradiol) has
occurred, we are able to show a significant inhibition of cell
growth and precursor incorporation which suggests a Ta
moxifen effect that may be independent of competition with
estradiol. This system also allows the investigation of hor
mone stimulation in an environment in which the specific
hormone studied can be eliminated for a fixed period of
time. With this system, we are able to see large stimulatory
effects of 17/3-estradiol and insulin as measured by thymidine incorporation in cells which are grown for 7 to 10 days
in IMEM-HS minus estradiol and insulin. The conversion of
cells from a stable nondividing state to exponential growth
is a much closer approximation of hormone-dependent
tissue than the relatively paltry stimulation usually observed
in culture. The attainment of these effects of hormones may
be important in allowing investigators to isolate specific
cellular products induced by hormone.
Interestingly, none of these hormonal components, either
alone or in any combination attempted by us, permits
growth of another human breast cancer cell line (MCF-7).
Finally, the ZR-75-1 cell line growing in IMEM-HS may
become a useful system to study the effects of single drugs,
drug combinations, and hormone and drug interactions in
human breast cancer. It provides a growing cell system,
free of serum contamination, for the study of drug interac
tion. It allows for the study of mechanisms of drug action
without the presence of nucleosides which are present in
serum (17). The system may be easily modified by the
addition of purines, pyrimidines, amino acids, and rescue
agents such as folinic acid in order to evaluate the mecha
nisms of drug action and drug toxicity. Furthermore, the
growth rate of the system can be altered by the omission of
a single hormone, allowing one to study the relationship of
drug action and growth rate.
The major problem with the system, thus far, lies in the
low plating efficiency. At present, the production of large
quantities of cells is not practical; however, in studying
hormone or drug interactions, this is not a problem, since it
is possible to plate the cells in charcoal-treated calf serum
and change to IMEM-HS as soon as the cells become
adherent. This low plating efficiency has also been noted in
many of the other cells lines that have been adapted to
1978
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
3827
J. C. Allegra and M. E. Lippman
defined medium. The HTC cells growing in IMEM-ZO re
ported by Thompson ef al. (19) have a plating efficiency of
0.1 to 1%. The HTC cells were fastidious in their require
ments for gentle handling and quality of medium. Burks
and Peck (4) also report a low plating efficiency for their
primary bone cell cultures in serum-free medium. They
found their plating efficiency to be density dependent, with
the cells exhibiting a low plating efficiency and a failure to
proliferate at a low density.
In summary, the ability to grow the ZR-75-1 human breast
cancer cell line in a defined serum-free medium supple
mented with hormones and transferrin will be advantageous
to the study of hormone interaction with human breast
cancer.
ACKNOWLEDGMENTS
We are grateful for the expert help of Le Esta Moran in preparing this
manuscript.
REFERENCES
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CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
VOL. 38
Cell Growth in Hormone-supplemented
Medium
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Fig. 1. ZR-75-1 human
growing continuously in
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Fig. 2. ZR-75-1 human
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220.
:
breast cancer cells. Cells
MEM plus 5% fetal calf
-
breast cancer cells. Cells
3 months in IMEM-HS. x
'
NOVEMBER
1978
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
3829
Growth of a Human Breast Cancer Cell Line in Serum-free
Hormone-supplemented Medium
Joseph C. Allegra and Marc E. Lippman
Cancer Res 1978;38:3823-3829.
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