[CANCER
RESEARCH 38, 4091-4100,
November
1978]
Unsaturated Fatty Acid Requirements for Growth and Survival of a Rat
Mammary Tumor Cell Line1
William R. Kidwell, Marie E. Monaco, Max S. Wicha, and Gilbert S. Smith
National
Cancer Institute,
Bethesda,
Maryland 20014
Abstract
A cell line, the growth and survival of which is markedly
affected by linoleic acid, has been established from a
carcinogen-induced
rat mammary tumor. The cells have
been continuously passaged in 5% rat serum plus 10%
fetal calf serum-supplemented
medium. The rat serum
component was found to be indispensable, for when it
was omitted the growth rate rapidly declined and the cells
died by 5 to 7 days. Removal of the rat serum from the
growth medium also resulted in a dramatic loss of Oil Red
O-positive droplets in the cells, suggesting that the lipid
component of rat serum might be a major growth-promot
ing principle in rat serum. This is likely since the total lipid
fraction, but not the delipidized protein fraction, could
largely supplant requirement of the cells for rat serum.
Pure linoleic acid was found to be effective in maintaining
the cell growth in delipidized serum or in whole fetal calf
serum-supplemented
medium. Fatty acid analysis re
vealed a 19-fold higher amount of linoleic acid in rat
serum than in fetal calf serum.
Although many properties of cells such as morphology
and agglutination (14), adenyl and guanyl cyclase activities
(10, 24), differentiation (34), membrane fluidity and cell
attachment (31), amino acid transport (18), and cloning
efficiency (12) are reportedly affected in specific instances
by essential fatty acids, the usual finding has been that cells
in culture show little impairment of division in the absence
of these fatty acids (for reviews see Refs. 1 and 16). The
essential fatty acids do, however, have pronounced effects
on growth and development in experimental animals (13)
probably at least in part via conversion to prostaglandins
(32) in addition to their structural role in cellular mem
branes.
There are indications that mammary cells may be espe
cially sensitive to lipids. Increased incidence of human
mammary carcinoma correlates positively with increased
consumption of animal fat (8), and a number of studies
have shown that high unsaturated fatty acid diets increase
the incidence of mammary adenocarcinomas in rats follow
ing DMBA2 administration (2-4). Also, the growth of transplantable mouse mammary adenocarcinomas is dramati
cally affected by dietary lipids (29). These effects have been
postulated to be due to an elevation of serum prolactin by
at the John E. Fogarty
International
Center Conference
Hormones and Cancer, March 29 to 31, 1978, Bethesda, Md.
2 The abbreviation used is: DMBA, 7,12-dimethylbenz(a)anthracene.
NOVEMBER
Materials and Methods
Cell Establishment. A small mammary tumor induced in
a Sprague-Dawley rat given 25 mg of DMBA at 50 days of
age was excised and minced in Eagle's minimal essential
medium containing 5% fetal calf serum and 2% collagenase
(type II; Worthington Biochemical Corp., Freehold, N. J.).
After incubation for 1 hr at 37°,the digest was filtered
Introduction
1 Presented
the dietary lipids (5) or to a suppression of immune surveil
lance because of a fatty acid-mediated inhibition of lympho
cyte proliferation (22).
In the present report we show that a cell line (WRK-1)
established from a DMBA-induced rat mammary tumor is
directly responsive to unsaturated fatty acids, linoleic acid
in particular. These results are not surprising in view of the
fact that mammary epithelial cells are embedded in an
adipose tissue matrix, with linoleic acid being one of the
most abundant fatty acids present in the rat gland. The
significance of the structural relationship of the glandular
component and adipose tissue is highlighted by the obser
vation that successful mammary tissue transplants require
mammary fat tissue as well as the epithelial component (7,
15).
on
through cheesecloth, and the filtrate was pelleted. The
pellet was washed with culture medium, and the cells were
resuspended and plated in Eagle's minimal essential me
dium containing 5% rat serum and 10% fetal calf serum
plus penicillin, streptomycin, and glutamine (19). After an
aliquot of the suspension was incubated in a Retri dish for
5 hr at 37°,the unattached cells and medium were trans
ferred to new Retri dishes and incubated for an additional
72 hr. Islands of epithelial-appearing cells were localized on
the dishes. Several of these were harvested by trypsinization with stainless steel rings. A successful culture of one
of these was established; the cells were cloned twice and
then maintained in culture for about 1 year with biweekly
culture splitting. From the time of initiation of the culture,
growth was maintained in Eagle's medium supplemented
with both rat and fetal calf sera.
Serum Preparation. Rat serum was prepared from ma
ture, nonpregnant, female rats of the same strain, with
most preparations consisting of sera pooled from 10 to 50
rats. The serum, collected from the abdominal aorta, was
heated at 56°for 30 min, sterilized by filtration, and stored
frozen at -20°.A single lot of fetal calf serum (Grand Island
Biological Co., Grand Island, N. Y.) was utilized for all of
the experiments reported here. Delipidization of sera was
performed with ethanoliacetone (30). For testing of serum
lipid fractions for growth promotion, the total serum lipids
[the material soluble in acetone:ethanol (1:1)] was taken to
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4091
W. R. Kidwell et al.
dryness by rotary evaporation and resuspended in a volume
of bovine serum albumin solution (1.4 g lipid-free albumin
in phosphate-buffered saline) equivalent to the original
serum volume. (Phosphate-buffered saline was prepared by
dissolving 8.0 g KCI, 1.15 g Na.HPO,, and 0.2 g KH.PO, in
1 liter of water.) The lipid suspension was then dialyzed for
24 hr against 400 volumes of phosphate-buffered saline (3
changes in the cold). The same bovine serum albumin
utilized to resuspend the lipid fraction was similarly dialyzed
and utilized as a control. The protein fraction recovered
from the acetone:ethanol precipitate was resuspended in
phosphate-buffered saline and dialyzed as described above.
Protein analysis (25) indicated that 66 to 72% of the serum
protein was recovered. The total serum lipid as quantitated
by H.jSO, charring (20) was recovered in 88% yield.
The lipoprotein fraction and alipoprotein serum were
prepared by adjusting the serum density to 1.25 g/ml with
solid Kl followed by flotation centrifugation (26).
Fatty Acid Analysis. Serum lipids extracted with
CHCI:i:methanol (3:1) were fractionated by thin-layer chromatography (21), and the appropriate fractions were sapon
ified. After acidification the liberated fatty acids were ex
tracted with hexane. The hexane extracts (about 2 ml) were
mixed with 1 ml of dimethylformamide and 20 /j.\ N,Ndiisopropylethylamine. The hexane layer was removed with
a stream of N,, and N. was bubbled through the dimethyl
formamide solution in the tube to remove residual amounts
of dissolved hexane. Then 2.3 mg of a-p-dibromoacetophenone were added, and the mixture was heated for 15
min at 65°.The bromphenacylated fatty acids produced
were chromatographed by high-pressure liquid chromatography on a Waters /¿C,*column with a gradient of 40 to
100% acetonitrile-water (17). Fatty acid derivatives were
detected by their absorption at 254 nm. Mass was estimated
by integration of peak areas with an HP 3380A integrator.
The mass is expressed in terms of crystalline bromphenacyllinoleate standard. When 0.01 or 25 /J.Qof labeled pal
mitic acid were utilized, recoveries of label in the respective
peak from the column were 88 and 97%, respectively. For
determination of the type of fatty acid synthesized in the
cells, 10 /J.Qof pure fatty acid carriers were added at the
time of lipid saponification. After derivatization the samples
were chromatographed. the peaks from the column were
collected, the solvent was evaporated off, and the radioac
tivity was quantitated.
Ultrastructure Studies. Cells grown in the presence of
fetal calf and rat serum were fixed in situ as monolayers
after reaching confluency. Fixation was accomplished with
1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH
7.2) for 30 min. After being rinsed in buffer, the monolayers
were postfixed in Dalton's chrome-osmium (6) for 1.5 hr,
dehydrated in ethanol. and infiltrated with and embedded
in a thin layer of Epon:Araldite. Following polymerization at
56°.the epoxy plastic layer with the intact monolayer was
stripped off the surface of the Petri dish. Appropriate areas
for ultrastructural evaluation were located by light micros
copy. These areas were cut out of the monolayer and
affixed to epoxy cylinders, and ultrathin sections were cut
on an 1KB Ultratome. Ultrathin sections were picked up on
Formvar-coated copper grids, stained with uranyl acetate
and lead citrate, and examined in a Siemens Elmiscop 102
4092
electron microscope at initial magnifications ranging from
3000 to 4000 diameters. The identical procedure was fol
lowed in studies where the effects of hormones on the
ultrastructural appearance of cells grown for 24 hr in
serum-free medium with or without added hormones.
Results
Cell Characterization. The cell line that has been isolated
has many features characteristic of secretory cells of rodent
mammary glands. Electron micrographie analysis (Fig. 1)
reveals intermediate tight junctions and gap junctions such
as those reported for mouse mammary glands (27, 28).
Under the influence of insulin, hydrocortisone, and prolactin (NIH B1), the cells take on an ultrastructural appearance
similar to mammary cells in early secretory phase (compare
Figs. 2 and 3). There is a marked development of the rough
endoplasmic reticulum with characteristic distended cisternae, the cytoplasm becoming filled with lipid droplets.
Hormone treatment results also in changes in surface
activity of cells as depicted by the increase in pinocytotic
vesicles, surface blebbing, and microvillar development
(Figs. 2 and 3). Compared to cells with the hormones
omitted, there is a development of an organized system of
cytoplasmic organelles which, except for the lipid droplets
and vesicles, appears to be due to a reorganization of
preexisting cytoplasmic components. Several features that
would make the mammary glandular epithelial origin more
certain were not found. These include the presence of
detectable amounts of «-lactalbumin or casein by sensitive
radioimmune assays or desmosomes.
In 2 respects the cells were similar to secretory cells of
the lactating gland. They accumulated massive amounts of
lipid droplets as detected by Oil Red O stain and contained
a hormonally responsive fatty acid synthetase. This is evi
dent from the lipid droplet accumulation in the hormonally
treated cells (Fig. 2). These droplets are most probably the
result of de novo lipid synthesis since ['"C]acetate incorpo
ration into fatty acids was stimulated 3- to 4-fold with insulin
Hormonal
stimulation
Table 1
of acetate incorporation
into fatty acids
Cells were grown to confluency in 60 HIM plastic dishes in fetal
calf and rat serum-supplemented
medium and then changed to
medium without serum. After 6 hr the medium was again replaced
(-serum).
Prolactin (NIH B1, 1 /xg/ml) and/or insulin (porcine,
from Lilly. 5 x 10 7 M) were added, and 30 hr later the cells were
pulsed for 2 hr with [14C]acetate (1 ¿iCi/ml; 2 x 10 5 M). The cells
were recovered from the plates by trypsinization,
and lipids were
extracted, saponified, derivatized, and separated by high-pressure
liquid chromatography.
Controls received no hormones.
W~lactindpm1,212185,1361,260938.55619,58410.44076,215%1.60.026.71.70.0150.625.
UHI
Ul
ControlFatty
acidLaurieLinolenicMyristicPalmitoleic-arachidonicLinoleicPalmiticOleicStearicTotal
dpmdpm15061.08026403.6962,1601,4648.820%1.70.0612.23.00.041.924.516.6Insulind
CANCER
RESEARCH
VOL. 38
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Fatty Acid Requirements of Mammary Tumor Cells
and 9-fold with insulin plus prolactin (Table 1). [The effect
of prolactin is likely due to a vasopressin contaminant (23).]
Neither hormone regimen markedly altered the relative
amount of synthesis of any particular size class of fatty acid
since the percentage of distribution of radioactivity in the
various fatty acids was essentially unchanged in the
presence or absence of hormones.
Evidence supporting the epithelial origin of the cells was
provided by the fact that considerable cell growth occurred
in medium in which L-valine was replaced by D-valine (9).
After a 24-hr lag, the D-valine-supplemented cultures grew
with a doubling time only one-third greater than that of
cells plated with L-valine (Chart 1).
Although we were unable to obtain tumors by inoculation
of the cells into nude mice, the cells appeared to be
transformed. They piled up in culture giving clear overlap
ping cellular edges (Fig. 4). Additionally, as has been noted
for some DMBA-induced mammary tumors, the cells con
tained rat leukemia virus (about 20 RNA copies/cell) and
the associated reverse transcriptase (H. Young, National
Cancer Institute, personal communication). The cells also
had an abnormal chromosome number as depicted in Table
2, where it is seen that the modal chromosome number is
80, or nearly double that of a diploid rat cell.
Rat Serum Requirement for Cell Growth and Survival. At
the time of initiating the tumor cells in culture and continu-
Chart 1. Growth of mammary tumor cells in medium containing D- or Lvaline. Cells were plated in medium with D-valine (93 mg/liter) or L-valine (46
mg/liter). The rat and fetal calf sera which were at 5 and 10% concentrations,
respectively, were dialyzed before use against 3 changes of Eagle's medium
with L-valine omitted (1000 volumes/dialysis).
Chromosome
ously thereafter, the cells have been maintained in medium
supplemented with 5% rat serum and 10% fetal calf serum.
Under these conditions the cells are filled with refractile
droplets that stain positively with Oil Red O indicating a
lipophilic nature. Within 24 hr of plating of cells in medium
with the rat serum component omitted, the lipid droplets
disappear (cf. Figs. 5 and 6). The cells assume a more
flattened appearance, and the number of cells per dish is
reduced. After 48 hr without rat serum, extensive pyknosis
and vacuolization of the cytoplasm are evident (Fig. 7).
After 5 days almost no viable cells are present in the dishes.
The rapid loss of lipid droplets when rat serum was
omitted suggested that the rat serum component necessary
for growth and survival might be a lipid. Consequently, the
total lipid fraction of rat serum and the protein fractions as
well were prepared and tested for their effects on tumor cell
growth in cultures supplemented with 10% fetal calf serum.
As shown in Chart 2, an amount of the total lipid fraction
equivalent to that present in 5% rat serum markedly stimu
lated cell proliferation compared to control cultures plated
in 10% fetal calf serum only. When both the rat serum
protein and lipid fractions were added back, the growth
was no better than with the lipid fraction alone. The total
rat serum protein fraction gave a modest stimulation of cell
growth, but it was considerably less active than the lipid
fraction (Chart 2).
With suitable adjustment of the density of the rat serum
with Kl and centrifugation. it was possible to remove the
very-low-density lipoproteins. the low-density lipoproteins,
and the high-density lipoproteins from serum. This alipoprotein serum contains the free fatty acids that are bound
to albumin. Each of the 2 fractions, the combined lipopro
teins and the alipoprotein serum fraction, were tested for
growth promotion as described in the last experiment. After
4 days of growth, the control cultures had increased in cell
number by about 50% (Table 3). The lipoprotein fraction,
which contained about 85% of the total serum fatty acids
(mono-, di-, and triglycérides and phospholipids), stimu
lated cell growth about 3-fold over the same time period,
while the alipoprotein serum fraction enhanced cell growth
rates by 8-fold. Both fractions together in amount equiva
lent to that present in 5% rat serum gave only a 9-fold
increment in growth rate, i.e., the removal of the lipoprotein
fraction had little effect on the growth-promoting effect of
rat serum. Oil Red O staining also showed that the accu
mulation of lipid droplets in the cells was almost totally
associated with the alipoprotein serum fraction.
A comparison of the free fatty acid profiles of rat and fetal
calf serum was made to see whether a difference in fatty
acid types or amounts might explain the rat serum require-
Table 2
number of WRK-1 cells
Subconfluent
cultures were treated for 5 hr with Colcemid (0.5 /¿g/ml). Mitotic cells dislodged
from the plates by shaking were pelleted,
swollen in 1% sodium citrate, and fixed with
methanohacetic
acid (3:1). The cells were placed on microscope slides, flamed, and stained with
0.1% méthylèneblue.
chromosomesNo.
No.of
ofcells751765771781791806811822830843850861871
NOVEMBER
1978
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4093
W. R. Kidwell et al.
Table 4
Free fatty acid concentrations of rat and fetal calf serum
Ã-¿g/ml
serum
acidLaurieLinolenicMyristicArachidonicLinoleicPalmiticOleicStearicRat4.00.77.23.6
Fatty
calf2.50.35.21.90.37.25.64.9Rat/fetal
calf1.62.31.41.918.62.3
23456
Table 5
DAYS
Chart 2. Effect of rat serum lipid and protein fractions on cell growth. The
cells were plated in 10% fetal calf serum with additions as indicated. O.
whole rat serum; •¿,
rat serum lipid fraction; O. rat serum protein fraction;
D, rat serum protein and lipid fractions; A, control buffer only.
Table 3
Effect of lipoproteins and lipoprotein-free rat serum on cell growth
Cells were plated at 5 x 104/dish in 10% fetal calf serumsupplemented medium to which the lipoprotein fraction of rat
serum or the alipoprotein serum was added. Controls received
phosphate-buffered saline against which the 2 serum fractions had
been dialyzed.
Addition
None
Lipoprotein
Alipoprotein serum
Lipoprotein + alipoproteinfree serum75,957
Cells/dish
±4,201"
126,735 ±4.368
437,922 ±2,516
468,472 ±8,078
" Mean ±S.D.
ment. As shown in Table 4, all the major fatty acids were 1to 3-fold higher in rat serum than in fetal calf serum.
However, the greatest difference was in linoleic acid which
was 19-fold higher in rat serum.
For assessment of the effect of linoleic acid on cell
growth and survival, cells were plated in delipidized serum
and various concentrations of linoleic acid were added. As
a control either no fatty acid or palmitic acid was added.
The results are presented in Table 5. Both the saturated and
unsaturated fatty acids were stimulatory and had concen
tration optima at about 0.5 ¿¿g/ml.
However, linoleic acid
was 5 times as effective as palmitic acid in stimulating
growth. Comparisons were also made of the relative effects
of several other fatty acids which were in higher concentra
tion in rat serum. As indicated in Table 6, linoleic acid was
more than 2 times as effective as were oleic, arachidonic,
or linolenic acids in promoting growth.
Calculations of the amount of linoleic acid that would be
contributed to culture medium supplemented with 10% fetal
calf or 5% rat serum (according to the data of Table 4)
indicate that the fetal calf serum supplement would contrib
ute 0.03 /*g/ml to the medium, while rat serum would
supply 10 times as much. The results of addition of pure
linoleic acid on the growth of the cells in medium contain
ing 10% whole fetal calf serum (Chart 3) indicate that
cellular growth rate is markedly improved by this fatty acid.
There is approximately a 4-fold increase in cells per dish
with the addition of linoleic acid. However, when whole rat
4094
Effect of linoleic and palmitic acids on cell growth
Cells were plated in medium containing 5% delipidized rat and
10% delipidized fetal calf serum. Two days later the cells were
harvested by trypsinization, and cell counts were performed. Pal
mitic acid at 5 ¿¿g/ml
was toxic to the cells. Data are expressed in
terms of the percentage of change in cell number over the initial
cell inoculation.
% increase in cell no.
tion (/¿g/ml)0
0.05
0.50
5.00Linoleic
acid54
acid51
57
83
52
203
180Palmitic
Table 6
Effect of unsaturated fatty acids on cell growth
The experiment was performed as described in Table 5 with the
fatty acids present at 1 /¿g/ml.
% increase in cell no.
Fatty acid
above control
375
126
141
154
Linoleic acid
Linolenic acid
Arachidonic acid
Oleic acid
40
T
o
X
I
52
O
30
20
10
+
01
«t
»r
oz
_J
UlUJ
DCK
yo
"-z
_1
+
Chart 3. Effect of pure linoleic acid and/or rat serum (flSi on cell growth.
FCS, fetal calf serum.
serum is added, linoleic acid has no further effect on cell
growth, indicating that most of the growth-promoting activ
ity of rat serum is due to the linoleic acid component.
Linoleic acid was not totally capable of replacing rat
CANCER
RESEARCH
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VOL. 38
Fatty Acid Requirements
serum insofar as growth was concerned (Charts 2 and 3).
One experiment performed with serum from hypophysectomized rats as compared to normal rats suggested that
some hypophyseal hormone might also have an effect on
cell growth. Medium supplemented with serum from intact
rats gave about 30% more growth than with serum from
hypophysectomized rats.
Conclusion
All cells of the mammary gland are present in a matrix
that is very rich in lipids. These lipids appear to be important
for glandular function and probably for growth as well,
based on the results of transplant experiments (7, 15).
Alterations of dietary lipid content, known to correlate
positively with the incidence of mammary cancer, may have
in addition to proposed secondary roles (5, 22) a direct
effect on tumor cell growth. This seems logical in view of
the fact that the types and amounts of fatty acids of the
gland are, to a first approximation, a reflection of the fatty
acids of the diet. However, there are indications from
nutritional studies that essential fatty acids are considerably
more important than other classes of fatty acids in the
physiology of the normal mammary gland (13). In this study
one such essential fatty acid, linoleic acid, was found to be
a major factor of rat serum for enhancing the amount of
growth and survival of a cell line established from a carcin
ogen-induced rat mammary tumor.
The WRK-1 cell line has been tested for a variety of
markers for identification purposes. No a-lactalbumin or
casein has been detected, nor are clearly defined desmosomes present. However, the lack of desmosomes does not
preclude the cells as being derived from epithelial compart
ment since these cellular structures are known to disappear
during certain functional stages of the mammary gland (27,
28). Additionally, there is present in normal glandular epi
thelium and in the tumors a second cell type, the myoepithelial cell, which does not have desmosomes. Both the
epithelial and myoepithelial cells possess tight and gap
junctions as do the WRK-1 cells (27, 28). The cells grow in Dvaline, a condition selective for epithelial cells (9), and they
have an epithelial morphology. The cells respond ultrastructurally to hormones as do the mammary epithelial cells
in expiant cultures (33). The presumptive response to prolactin is probably due not to the prolactin molecule but to
vasopressin, a contaminant in the prolactin preparation
(23). Whether this indicates a physiological role of vaso
pressin for mammary cells remains to be seen.
Like mammary cells of lactation (11), there is a large
stimulation of acetate incorporation into fatty acids by
insulin plus the prolactin preparation. The electron micrographic analysis presents strong evidence that hormonal
treatment produces an actual increase in fatty acid synthe
sis rather than affecting only [14C]acetate transport, since
the treated cells but not the controls are filled with lipid
droplets. These droplets cannot be a consequence of up
take of lipids from the growth medium because the experi
ments are performed in serum-free medium.
While there is some uncertainty as to the origin of our
cell line, the probability is that it represents a tumor cell type
that became established in culture because its high linoleic
NOVEMBER
of Mammary
Tumor Cells
acid requirement was met by the rat serum supplement in
the culture medium. High concentrations of essential fatty
acids may be a general requirement of mammary cells of all
types, since we have recently demonstrated that primary
cultures of mammary gland alveolar cells and those from
DMBA-induced rat tumors show a marked stimulation of
growth upon the addition of linoleic acid to the growth
medium. Additionally, we have demonstrated that the mam
mary gland selectively alters the ratio of linoleic acid relative
to palmitic acid when the gland is induced to proliferate by
perphenazine administration.3 These results point to a need
for a great effort to understand growth and differentiation
as consequences of the coordinated and integrated re
sponse of various cell types of the mammary gland to
hormones.
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1978
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23 Monaco. M. E., Lippman. M E . Knazek. R., and Kidwell. W. R.
Vasopressin Stimulation of Acetate Incorporation into Lipids in a Dimethylbenz(a)anthracene-induced Rat Mammary Tumor Cell Line. Cancer Res.,38. 5001-5004. 1978.
24. Orly. J.. and Schramm, M. Fatty Acids as Modulators of Membrane
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Contacts in the Mouse Mammary Gland 1 Normal Gland in Postnatal
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4096
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31. Schaeffer, B. E.. and Curtiss. A. S. G. Effects on Cell Adhesion and
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32. Van Dorp, D. A., Beerthius. R. K., Nugterien. D. H.. and Vonkeman. H.
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34. Weeks. G. The Manipulation of the Fatty Acid Composition of DictyosteHum Discoidium and Its Effect on Cell Differentiation. Biochim. Biophys.
Acta. 450: 21-32. 1976.
CANCER
RESEARCH
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VOL. 38
Fatty Acid Requirements
of Mammary
Tumor Cells
•¿
'
mm.
Fig. 1. Junctional complexes of the eelIs. Upper arrow, gap junction; center arrow, area of intermediate and tight (unctions, x 35.000. Inset, tight junction
between apposing cells, x 140,000
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1978
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W. R. Kidwell et al.
W
''is
A*"-.
r.
r
1
l
,^
Fig. 2. Hormonal effects on cell ultrastructure. Cells treated for 24 hr with insulin (5 x 10 7M), prolactin (B1,1 n9/m|). and hydrocortisone (5 x 10"" M) in
medium with serum omitted. R, distended rough endoplasmic reticulum; P, pinocytotic vesicles; L, lipid droplets; M, mitochondria, x 14,000.
4098
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VOL. 38
Fatty Acid Requirements
of Mammary
Tumor Cells
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Fig. 3. Ultrastructure of cells in the absence of hormones. Note the absence of lipid droplets, the relative scarcity of mitochondria, and the lack of
distension of the cisternae of the rough endoplasmi reticulum.
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1978
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W. R. Kidwell et al.
Fig. 4. Abnormal piling up of the cells in culture. Note the prominent overlapping of cellular edges.
Fig. 5. Accumulation
of lipid droplets in cells grown in the presence of both rat and fetal calf serum. The lipid droplets
the cytoplasm. Oil Red 0.
Fig. 6 Absence of Oil Red O-staining material in cells grown for 24 hr with the rat serum component omitted.
Fig. 7. Extensive pyknosis
4100
and vacuolization
appear as discrete
foci throughout
of cells 48 h r after plating in medium with rat serum omitted.
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VOL. 38
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Unsaturated Fatty Acid Requirements for Growth and Survival
of a Rat Mammary Tumor Cell Line
William R. Kidwell, Marie E. Monaco, Max S. Wicha, et al.
Cancer Res 1978;38:4091-4100.
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