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[CANCER RESEARCH 30, 127-132,
January 1970]
Expression of Differentiated Function by Mammary Carcinoma
Cells in Vitro1
Roger W. Turkington and Marie Riddle
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27706, and the Division of Endocrinology,
Administration Hospital, Durham, North Carolina 27705
SUMMARY
Expiants of C3H mouse and rat R3230AC mammary
carcinomas were cultured
in the presence of insulin,
hydrocortisone, and prolactin in order to test the ability of
these cells to differentiate into cells producing specialized cell
products. Although normal epithelial cells differentiate into
alveolar secretory cells which synthesize casein, a-lactalbumin,
and lactose synthetase A protein, C3H carcinoma cells fail to
produce significant increases in these differentiated functions
under the in vitro conditions. This failure is unrelated to rapid
proliferation since the C3H carcinoma cells proliferate at a rate
similar to that of normal cells and the rapidly proliferating
R3230AC carcinoma cells can markedly increase casein
synthesis in response to hormonal stimuli in vitro. The results
support the concept that cells which form C3H tumors cannot
limit the cell population size through the normal sequence of
cell differentiation.
INTRODUCTION
The formation of a tumor by some neoplastic cells would
appear to result from the high rate of cell division among such
cells. It is recognized however that many types of cancer cells
divide less often than some normal cells (1, 9, 15). One
possible explanation
for tumor formation
by slowly
proliferating cells could lie in the potential inability of such
cells to differentiate.
While normal
populations
of
continuously proliferating cells are constantly renewed by
frequent stem-cell division, the size of such cell populations is
limited by the production of daughter cells which can
synthesize highly differentiated
cell products and which
seldom divide. Although specialized cell products may be
formed by some cancer cells, many types of neoplastic cells
exhibit little functional evidence of differentiation. These
latter cells are often designated "dedifferentiated"
cells, but
no evidence exists that they have in fact lost the capacity to
synthesize specialized proteins.
In order to test neoplastic cells for their capacity to
synthesize specialized proteins we have studied milk protein
'This work was supported by USPHS Grant CA-10268 from the
National Cancer Institute, NIH.
Received March 6, 1969; accepted May 21, 1969.
Veterans
synthesis by mammary carcinoma cells. Previous studies
established that normal mouse mammary epithelial cells form
casein, a-lactalbumin, and the A protein of lactose synthetase
in response to specific hormonal stimuli. Epithelial cells which
divide in the presence of insulin and hydrocortisone in organ
culture produce daughter cells which can synthesize these milk
proteins in response to stimulation by prolactin and insulin
(22, 24, 26). Cells formed in the presence of insulin alone do
not express such new genetic information. Since mammary cell
differentiation is dependent upon hormonal stimulation the
ability of mammary carcinoma cells to differentiate can be
assayed by allowing these cells to grow in the requisite
hormonal environment.
MATERIALS AND METHODS
Organ Cultures.
The carcinomas
studied were the
spontaneous,
virus-associated carcinoma in the C3H/HeJ
mouse and the R3230AC transplantable carcinoma in the
Fischer rat. Lactational,
midpregnancy,
and neoplastic
mammary tissues were prepared and incubated in Medium 199
(Microbiological Associates, Bethesda, Md.) as previously
described (23). Each hormone was present in the medium at a
concentration of 5 jug/ml.
Casein Synthesis. The rate of casein synthesis was measured
by allowing the expiants to incorporate 32P (carrier-free, 60
¿iCi/mlmedium) or 14C-labeled amino acids (a mixture of 20
amino acids, 10 juCi/ml medium) during a 4-hr labeling period.
The labeled casein was isolated from the 105,000 X g
supernatant of tissue homogenates and culture medium by
precipitation with rennin and calcium ions in the presence of
authentic C3H/HeJ mouse or Fischer rat casein "carrier," and
prepared for polyacrylamide gel electrophoresis as described
previously (24). A lower gel formed with 7.5% acrylamide and
8 M urea and buffered at pH 8.83 with 0.375 M Tris-HCl
provided optimal resolution of each of the components of
casein from these 2 species. Potassium persulfate (50 mg/100
ml) and riboflavin (0.5 mg/100 ml) were present as initiators,
and a minimal amount of tetramethylethylenediamine
was
used as accelerator. The upper gel was 5% acrylamide and 8 M
urea, and other buffer systems were those of Jovin et al. (IO).
The casein preparations were subjected to electrophoresis at
10 ma/tube in a temperature-regulated apparatus at 25°.The
gels were then fixed in 7.5% acetic acid, stained with Amido
black, and sectioned with a manual slicing device (5) which
divided the gels from the origin into uniform slices 1.0 mm
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127
Roger W. Turkington and Marie Riddle
thick. The gel sections were then counted in a liquid
scintillation spectrometer with the use of Bray's solution (2).
Lactose Synthetase. Galactosyltransferase
reactions of the
lactose synthetase enzyme system were assayed by the
radioactive method described earlier (22) except that uridine
triphosphate was present in the assay tubes at a concentration
of 2 mM. The A protein galactosyltransferase
activity is
measured by the rate of galactose-14C transfer from uridine
diphosphogalactose to TV-acetylglucosamine. a-Lactalbumin (B
protein) modifies the substrate acceptor specificity of this
enzyme to include glucose (3) and a-lactalbumin activity is
assayed by the rate of galactose-14C transfer to glucose in the
presence of an excess of exogenous purified bovine A protein.
Other Methods. DNA in the tissues was measured by the
method of Burton (4). Cytoplasmic noncasein phosphoprotein
was measured by the method of Juergens et al. (11). For
measurement of the rates of total cytoplasmic protein, tissues
were cut into small expiants and exposed to Medium 199
containing 14C-labeled amino acids (10 /¿Ci/ml)for 3 hr.
14C-Labeled proteins were precipitated from the 1000 X g
supernatant with 5% trichloroacetic acid. After hydrolysis of
nucleic acids in trichloroacetic acid at 90° for 15 min the
precipitates were washed repeatedly with trichloroacetic acid
and ether-ethanol, dissolved in 5 M acetic acid, and counted in
Bray's scintillation fluid.
RESULTS
Chart la shows the electrophoretogram radioactivity profile
of casein-32? isolated from mammary expiants of a 10-day
lactating Fischer rat, the strain of origin of the R3230AC
carcinoma. The electrophoretic mobilities of the 9 radioactive
peaks observed are identical with those of the stained bands of
authentic Fischer rat casein. Similar peaks of casein-32? are
formed in small amounts by differentiated midpregnancy
epithelial cells incubated in medium containing insulin (Chart
1ft), and, as shown previously (24), a marked stimulation of
the synthesis of these casein components occurs in tissue
which differentiates in the IFF2 medium. Radioactive peaks
representing the synthesis of specific casein phosphoproteins
by R323OAC cells incubated on medium containing insulin
are shown in Chart le. The rate of incorporation of
P into
casein per cell, based upon the peak values in each component,
is approximately 10% that of the lactational tissue. The rate of
synthesis of casein-32? components in tumor cells derived
from a lactating donor was approximately 25% of the rate of
the mammary cells of the donor. "Chase" experiments
revealed no detectable turnover of 32P-labeled casein in any of
the tissues studied, indicating that the amount of casein-32?
recovered after the 4-hr labeling period reflects the rate of
casein formation. Culture of the carcinoma cells in the IF?
medium for 48 hr results in a 200% increase in the synthetic
rates, and the relative rates of synthesis of the casein
components are similar to those observed in the normal
2The abbreviation
prolactin medium.
128
used
is: IFF,
insulin,
hydrocortisone,
and
tissues. That is, although the rate of synthesis of each
component is below that in the normal cell, the neoplastic cell
maintains the same radioactivity profile or ratio of synthesis
among the components which is characteristic of the normal
cell.
Chart 2a shows the characteristic
electrophoretogram
radioactivity profile of casein-32? synthesized by lactational
C3H/HeJ mouse mammary gland. Chart 2b illustrates a similar
stimulation of casein synthesis as a concomitant of cell
differentiation
in the IF? medium. Synthesis of casein
components can be detected in C3H/HeJ carcinomas, as shown
in Chart 2c, but the rate of synthesis per cell is markedly
below that of the lactational tissue. The radioactivity in the
major casein bands (Gel Sections 10 to 27) ranges between 1
and 3% of the lactational values. Although growth in the IF?
medium results in slight stimulation of the 3 slowest com
ponents, there is no significant stimulation of the peaks
contained in Gel Sections 10 to 27, which represent the major
proteins characteristic of the differentiated cell (cf. Chart 2a).
Inclusion or omission of hormones in the medium just during
the 4-hr labeling period did not alter the results shown. Results
similar to those depicted in Charts 1 and 2 were also obtained
with 14C-labeled amino acids as the radioactive precursor.
Another differentiated function, lactose synthesis, was also
compared in normal and neoplastic mammary cells by
measuring the activity of the terminal and rate-limiting
enzyme in the biosynthetic pathway, lactose synthetase.
Previous studies (22) demonstrated that both the A protein
(galactosyltransferase)
and the B protein (a-lactalbumin) of
this enzyme system are induced in cells which differentiate in
response to insulin, hydrocortisone, and prolactin. Table 1
shows that the A and B protein activities are present in both
carcinomas, but the cellular levels are V15 to Vso of those
found in the corresponding differentiated cells. Table 2
compares the capacity of normal and neoplastic mammary
epithelial cells for hormone-dependent differentiation in terms
of the formation of the lactose synthetase proteins. As a
consequence of cell division in the IFF medium the A and B
protein activities of normal tissues rise 500 to 600% above
those observed in expiants incubated on medium containing
the single hormone insulin. Although some rise in enzyme
activity was observed in the carcinomas incubated on IFF
medium, this was only an approximately
50% increase.
Experiments were performed in which extracts of the
neoplastic and normal cells were mixed and assayed. The
results demonstrated
that these differences could not be
attributed to the presence of an enzyme inhibitor in the
neoplasms or to the presence of an activator in the normal
cells.
In order to determine whether the differences in rates of
casein synthesis and in lactose synthetase activity observed
between carcinoma and normal cells could relate to
corresponding differences in rates of cytoplasmic protein
synthesis in general, several other incorporation studies were
carried out. The results shown in Table 3 are representative of
3 such experiments. In terms of the rates of 14C-labeled amino
acid incorporation into total cytoplasmic protein, it can be
seen that the markedly reduced rates of milk protein
formation in the carcinoma cells cannot be explained by the
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Mammary Carcinoma Cells in Vitro
Table 1
Table 3
Activities of the A and B proteins of lactose synthetase
in lactational mammary gland and mammary carcinomas
Rates of synthesis of total cytoplasmic protein
and cytoplasmic noncasein phosphoprotein in expiants
of lactational mammary gland and mammary carcinomas
The values are the mean ±l S. D. of replicate determinations on 6
tissues in each group. The A and B protein activities were assayed
separately in tissue homogenates using the radioactive assay of Brew
et al. (3) as described previously (15). The neutral sugar product
formed in the tissue homogenates in the presence of uridine diphosphogalactose-14C, glucose, and exogenous purified bovine A protein
was shown by paper chromatography to be lactose.
Lactose synthetase
product/min/20 ¿igDNA)
A protein
Fischer rat
Mammary gland
carcinomaC3H
R3230AC
mouse
Mammary gland
Carcinoma2.99
B protein
Noncasein phosphoprotein
Fischer rat
Mammary gland
R3230AC carcinoma
1250
2360
918
868
C3H mouse
Mammary gland
Carcinoma
3640
2510
875
600
±0.42
0.052
±0.0183.66+0.97 In relation
±1.17
0.292 ±0.0952.45
0.120+0.036
to these observations on rates of milk protein
synthesis, studies on concurrent rates of cell proliferation were
performed. Table 4 lists indices for labeling of normal and
neoplastic cells with thymidine-3H. It was shown previously
Effect of various hormones on lactose synthetase
activity in expiants of midpregnancy mammary
glana ana oj mammary carcinomas
Expiants were incubated on Medium 199 containing insulin or IFF
for 48 hr, and were then weighed and assayed for enzymatic activity
as previously described (15). Since a large proportion of the mam
mary gland explant weight represents fat cells, only relative increases
in response to hormones are compared in this experiment. The results
are representative of 3 such experiments.
Fischer rat
Mammary gland
Total protein
±0.48
±0.0905.55
0.216
Table 2
Tissue
Radioactivity
DNA/3 hr)
Hormone
system
Insulin
IFF
Lactose synthetase
(mamóles product/min/mg tissue)
A protein
B protein
0.007
0.032
0.0120.004
DISCUSSION
The important studies previously reported by Huggins et al.
(8), Kim and Furth (12-14), and others indicated that several
0.003
0.018
carcinomaC3H
R3230AC
IFFInsulin
that these indices correlate with mitotic indices and thus are a
reflection of different rates of cell proliferation rather than
merely different rates of uptake of the radioactive precursor
(23). Significant numbers of epithelial cells in the developing,
midpregnancy gland are in the S period at the beginning of
organ culture and, as shown previously, many cells are induced
to synthesize DNA in response to the presence of insulin in the
medium (11,12). Very few of the highly differentiated cells of
lactational tissue are proliferating. Of particular note was the
observation that the C3H carcinoma cells proliferated no
more rapidly than normal midpregnancy cells. In contrast
R3230AC carcinoma cells proliferated most rapidly, with all
cells initiating DNA synthesis during the first 24-hr period.
0.0110.002
mouse
Mammary
glandCarcinomaInsulin
IFFInsulin
0.0450.028
0.0150.004
IFF0.010
0.0400.008
0.005
100% increase in the R323OAC cells or the 50% reduction in
the C3H carcinoma cells as compared to normal cells. The rate
of incorporation
of 32P into another population
of
cytoplasmic proteins, the noncasein phosphoproteins, was also
not sufficiently different between the normal and carcinoma
cells as to account for the marked differences observed in
casein phosphoprotein synthesis.
Table 4
Labeling indices of mammary epithelial cells and carcinoma cells
during organ culture
Expiants were exposed to Medium 199 containing tritiated
thymidine (0.5 »jCi/ml)for the indicated periods of culture. The
preparation of autoradiographs and determination of the percentage
of cell nuclei labeled was previously described (16).
Tissue
Epithelial
4 hr cells labeled
~^f hr (%)
Fischer
ratMidpregnancy
glandLactationalmammary
glandR3230AC
mammary
carcinomaC3H
mouseMidpregnancy
glandLactationalmammary
glandCarcinoma70.82460.74351004034
mammary
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129
Roger W. Turkington and Marie Riddle
12,500
1A
10,000
1B
7,500
1500
5,000
1000
2,500
500
1C
1500
o IOOO
5
10
15 20 25 30
GEL
SECTION
35
è 500
5
IO
IS
20
25
CELSECTION
30
IO
35
15
20
25
30
35
GELSECTION
Chart 1. Electrophoretogram radioactivity profiles of casein components labeled with
P in vitro by mammary tissues from Fischer rats, a,
lactational tissue, labeled 0 to 4 hr on Medium 199 containing insulin, b, midpregnancy tissue culture for 44 hr on Medium 199 containing insulin
(o—o) or insulin, hydrocortisone, and prolactin (•—•)
and labeled during the 44- to 48-hr period. Since fat cells comprise a large proportion of
these expiants, only the relative response per mg tissue is represented, c, carcinoma expiants incubated as in Chart Ib.
2000
20,000
800
2C
1500
600
400
IL
o 200
500
15 20
25
GELSECTION
30
35
5
10
15 20 25
GELSECTION
30
35
15
20
25
30
35
GELSECTION
Chart 2. Electrophoretogram radioactivity profiles of casein components labeled with 32t P in vitro by mammary tissues from C3H mice.
Experiments were performed as in Chart 1. a, lactational tissue; b, midpregnancy tissue; c, carcinoma tissue.
types of mammary carcinomas in rats are hormone-depen
dent, and that often those hormonal conditions which are
most effective in inducing mammary gland growth or secre
tion correlate with increased rates of tumor growth in vivo.
In contrast the growth patterns of mammary carcinomas in
mice have often appeared to be independent of hormonal
factors (6). The hormonal dependence or responsiveness of
mammary carcinoma ecus may however be more variable
than could be predicted from the species of origin alone.
Previous studies on the R3230AC carcinoma of the Fischer
rat have demonstrated
that its cells can induce DNA
polymerase, initiate DNA synthesis, and subsequently divide
independently of insulin, which is required for a stimulation
130
of these processes in normal Fischer rat mammary epithelial
cells (23, 26). Although cell growth in the Fischer rat
mammary gland in vitro is profoundly altered by estrogenic
hormones it is unaltered by estrogens in the R3230AC
carcinoma in vitro (23). In contrast DNA synthesis and cell
growth in C3H mouse carcinomas in vitro are modified by
insulin and estrogenic hormones in a manner indistinguish
able from that in the normal mouse mammary gland (23,
26).
The purpose of these studies was to determine another aspect
of hormonal responsiveness in these 2 model tumor systems,
namely their capacity for hormone-dependent cell differentia
tion. In contrast to almost all other tissues of the body, the
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Mammary Carcinoma Ceils in Vitro
HORMAL
observed in the rates at which the normal and neoplastic cells
incorporate 14C-labeled amino acids into total cytoplasmic
protein
or 32P into cytoplasmic noncasein phosphoprotein do
DIFFERENTIATED
LIFESPAN
Chart 3. Scheme of cell proliferation and differentiation in normal
and neoplastic mammary cells of the C3H mouse.
chemical nature of the molecular inducers of cell differentia
tion in the mammary gland are known. In vitro they are
insulin, hydrocortisone,
and prolactin (11, 21), hormones
which cause morphological differentiation
and selectively
induce milk proteins (22, 25). The milk proteins which have
been assayed in these experiments represent specific proteins
which normally are synthesized uniquely by differentiated
mammary secretory cells. During the hormone-dependent
development of the normal glandular tissue the onset of
synthesis of these proteins represents the expression of new
genetic information, and thus they serve as markers of
regulation of specific gene activity (21, 25). Casein is a group
of phosphoproteins
which are precipitated selectively by
rennin and calcium ions. All of the material so isolated from
the normal tissues has previously been shown to be
electrophoretically
identical with authentic Fischer rat or
C3H/HeJ mouse casein (24, 27). The phosphoproteins thus
isolated from the neoplastic tissues also consist of components
whose electrophoretic
mobilities are identical with those
authentic caseins of their strain of origin, both by protein
staining and by radioactivity patterns. a-Lactalbumin in the
R323OAC carcinoma has been further characterized by the
electrophoretic
mobility of the 14C-labeled protein (27).
Earlier studies by Hilf (7) on fluid in the R3230AC carcinoma
lend further confidence to the interpretation
that these
specific milk proteins were synthesized in vitro. Hilf reported
the presence of casein components in the "milklike" tumor
fluid, and Hilf (7) and Shatton et al. (16) reported the
presence of low concentrations of lactose in the fluid.
The neoplastic cells studied in the present experiments are
characterized by a marked reduction in the rate of formation
of casein and in the cellular levels of lactose synthetase A
protein
and a-lactalbumin
in comparison
to normal
differentiated cells. The differences in protein synthesis relate
to specialized milk proteins rather than to differences in rates
of total protein synthesis. As shown in Table 3 the differences
not in themselves account for the much greater differences in
rates of milk protein formation which were observed.
An increase in the level of differentiated function in the C3H
mouse mammary gland is dependent upon cell division
(18-20). During the 48-hr incubation period approximately
70% of the epithelial cell nuclei become labeled with tritiated
thymidine (17). Undifferentiated cells as a result of hormonal
stimulation produce daughter cells which can synthesize
specific milk proteins. As shown in Table 4 differentiated
lactational cells seldom divide. The proportions of C3H
carcinoma cells and midpregnancy mammary epithelial cells
which divide during the 48-hr incubation period are similar, a
finding which is consistent with the low rates (relative to some
other tumors) of cell proliferation in vivo reported by
Mendelsohn et al. (15). During each 24-hr period 100% of the
R3230AC carcinoma cells initiate DNA synthesis (23), a rate
of proliferation which greatly exceeds that of the midpregnancy epithelial cells. R3230AC carcinomas are able to
increase their rate of casein synthesis, but milk protein
formation is not associated with decreased rates of cell
proliferation. The low synthetic rate per cell for casein may on
the other hand be viewed as reflecting the direction of more
biosynthetic activity toward the processes of cell division. The
rate of proliferation per se would not appear to preclude the
expression of differentiated function in C3H carcinoma cells
however. On the contrary their failure to differentiate may
represent a factor in the abnormal growth potential of these
cells. This pattern of cell proliferation is shown schematically
in Chart 3. Proliferation of normal epithelial stem cells in IFF
medium involves the formation of differentiated secretory
cells which, because they seldom divide, limit the size of the
cell population (it is not known whether such cell divisions
result in the formation of 1 or 2 differentiated cells). The
experimental evidence for this model has been previously
reviewed (21). Since division of C3H mammary carcinoma
cells in the IFF medium fails to produce significant numbers of
new, fully differentiated, nondividing cells, daughter cells
which are produced may retain the capacity of stem cells to
divide into 2 new daughter cells, thus leading to the formation
of a large aggregate of undifferentiated mammary cells. The
low level of differentiated function in the 2 carcinomas may
reflect abnormally low rates of milk protein synthesis in a
majority of each cell population, or fully differentiated
function in a small proportion of the cells. Studies to
distinguish between these 2 possibilities are in progress. It is
possible that various tumors or various stages of tumor growth
may fall into at least 2 classifications: (a) cells which grow
more rapidly than normal cells; and (b) cells which cannot
limit the size of the cell population by differentiation. Because
the molecular inducers of alveolar differentiation of mammary
cells are known to be specific hormones, it has been possible
to test experimentally the capacity of cells which form C3H
carcinomas to differentiate. Although other factors may be
important in determining the neoplastic character of the C3H
carcinoma cell the present evidence is consistent with the
concept that defective mechanisms for hormone-dependent
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131
Roger W. Turkington and Marie Riddle
differentiation
may be a basic abnormality
mammary carcinoma cell.
of the C3H
ACKNOWLEDGMENTS
We thank Drs. Ian Trayer and Robert L. Hill for a generous gift of
purified bovine lactose synthetase A protein.
14.
15.
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CANCER RESEARCH VOL. 30
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1970 American Association for Cancer Research.
Expression of Differentiated Function by Mammary Carcinoma
Cells in Vitro
Roger W. Turkington and Marie Riddle
Cancer Res 1970;30:127-132.
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