(CANCER RESEARCH 43. 3348-3357,
July 1983]
Alterations of Human Endometrial Stromal Cells Produced by N-MethylN '-nitro-Af-nitrosoguanidine1
B. Hugh Dormán,2Jill M. Siegfried,3 and David G. Kaufman4
Department of Pathology [B. H. D., D. G. K.] and Cancer Research Center [J. M. S., D. G. K.], University of North Carolina, Chapel Hill, North Carolina 27514
Stromal cell cultures obtained from human endometrium were
treated repetitively with A/-methyl-A/'-nitro-A/-nitrosoguanidine in
cells to survive in culture and their apparent resistance to the
effects of carcinogens have made it difficult to study their trans
formation by chemical agents. Recently, Milo et al. (28) have
reported that human foreskin epithelial cells treated with MNNG5
vitro at concentrations ranging from 0.5 to 4.0 ng¡m\,and alter
ations in growth potential and morphology were analyzed. A
single exposure to the carcinogen resulted in morphological
evidence of toxicity and reductions in growth rates, plating
efficiency, and saturation density as compared to solvent-treated
and other carcinogens acquired the ability to grow in soft agar
and to invade chick embryonic skin in vitro.
We were interested in studying chemical transformation in
normal human endometrial stromal cells. These cells are capable
of long-term survival in culture and retain some of the in vivo
control cells. Cytotoxicity was reduced after additional exposures
to the carcinogen. Following repetitive treatments with A/-methylN '-nitro-W-nitrosoguanidine, human endometrial Stromal cells de
features of the parent tissue. These properties would permit a
spectrum of biological characteristics to be compared in normal
and carcinogen-treated cells during the course of transformation
in vitro. Human endometrial cell cultures have not been utilized
previously in studies of chemical carcinogenesis in vitro; there
fore, it is valuable to consider some of the properties of these
cell cultures. We have developed methods for culturing human
endometrial stromal cells, the predominant cell type of the en
dometrium (40), and have documented the apparent biological
normalcy and limited life span in vitro of these cultures, averaging
16 to 18 passages before senescence (13). These cells have
been identified as stromal in origin based on light and electron
microscopy (13). Stromal cells act as the supportive tissue for
the glandular epithelium, but they are distinguishable from fibro
blasts which are also present in the endometrium. They also
function to prepare the endometrium for implantation of the
blastocyst. During the menstrual cycle and pregnancy, stromal
tissue undergoes many morphological and biochemical changes
which are indicative of its capacity for differentiation. We have
documented that, in culture, human stromal cells respond to
steroid hormones by aggregating and producing PAS-positive
material, presumably glycogen (13), as occurs in vivo. In humans,
endometrial stromal cells are renewed regularly following men
struation; thus, they possess many characteristics which may
contribute to the development of tumors in vivo and provide an
interesting target for transformation studies.
ABSTRACT
veloped enhanced growth potential, the capacity to form mac
roscopic colonies in soft agar, and elevated 7-glutamyltranspeptidase activity. Carcinogen-treated cells displayed atypical mor
phology characterized by irregularities in cell and nuclear size
and shape, large bizarre nucleoli, increased nuclearcytoplasmic
ratios, and cellular crowding. Control cells did not display altered
morphology or growth parameters even following multiple ex
posures to solvent and repetitive subculturing. These alterations
in growth potential and morphology suggest that the cells are
progressing towards preneoplastic and perhaps neoplastic trans
formation in vitro.
INTRODUCTION
Little is known about the alterations which take place in human
cells following exposure to chemical carcinogens. While many
studies have documented malignant transformation in both fibroblasts (7, 12, 24) and epithelial cells (17, 21, 25, 33, 38) from
experimental animals, the change from a cell with a normal
phenotype to a tumor-producing cell has rarely been successful
with cultured human cells. Transformation studies have utilized
human fibroblasts, some of which were derived from tumors or
from tissues genetically predisposed to cancer (4, 20, 32, 35).
Human fibroblasts from foreskin and lip have also been trans
formed in vitro with chemical carcinogens (23, 27, 37). In these
studies, cells acquired alterations, such as increases in saturation
density and growth in soft agar, similar to the progressive
changes reported in cells derived from experimental animals (3,
15, 26). Since carcinomas are the most prevalent tumor in
humans, epithelial cells are considered an important target for
transformation studies. The limited ability of human epithelial
1This work was supported by Contract N01-CP75956 and Grant CA31733 from
the National Cancer Institute.
2 Recipient of a predoctoral scholarship from the Chemical Industry Institute of
Toxicology. Present address: Chemical Industry Institute of Toxicology, 6 Davis
Drive, Research Triangle Park. N. C.
3 Recipient of a postdoctoral fellowship (CA07058) from the National Cancer
Institute
4 Recipient of Research Career Development Award CA00431 from the National
Cancer Institute. To whom requests for reprints should be addressed.
Received August 16.1982; accepted April 11,1983.
3348
MATERIALS AND METHODS
Cell culture methods for establishment of endometrial stromal cell
cultures from hysterectomy specimens have been described previously
(40). Briefly, monolayer cultures were grown in CMRL Medium 1066
supplemented with 10% heat-inactivated fetal bovine serum, 10 mw Lglutamine, streptomycin (100 ^g/ml), penicillin (100 units/ml) (all obtained
from Grand Island Biological Co., Grand Island, N. Y.), 25 mn/i W-2hydroxyethylpiperazine-/v"-2-ethanesulfonic
acid (Sigma Chemical Co.,
St. Louis, Mo.), and insulin (4 ^g/ml) (crystalline bovine, 24 ID/mg; Sigma
Chemical Co.). This medium was used to culture cells in all experiments
unless otherwise noted. The human stromal sarcoma cell line was
5The abbreviations used are: MNNG. W-methyl-AT-nitro-N-nitrosoguanidine;
PAS. periodic acid-Schiff's reagent; CMRL, Culture Medium Research Laboratory;
GGT, 7-glutamyl transpeptidase;
DES, diethylstilbestrol.
CANCER
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Effects of MNNG on Human Endometrial Cells
established by the same techniques used for normal endometrial tissue
from an invasive pelvic lesion diagnosed as endometrial stromal cell
myosis. Cultures were periodically screened for Mycoplasma contami
nation using the Hoechst staining technique (6) and were found to be
negative.
Carcinogen Treatment and Analysis of Growth Characteristics. At
subconfluent density (1.5 x 104 cells/sq cm), stromal cell cultures were
placed in Hanks' balanced salt solution at 37°and treated for 30 min
adult animals was irradiated with 450 rads (cobalt source) 24 hr prior to
cell injection.
with MNNG (Aldrich Chemical Co., Milwaukee, Wis.) dissolved in spectral
grade acetone (Fisher Scientific Co., Pittsburgh, Pa.). MNNG was stored
as a powder at -70°; solutions were freshly prepared and used imme
of endometrial stromal cells with MNNG resulted in measurable
cytotoxicity as analyzed by growth inhibition. Decrease in colonyforming ability was not used to monitor cytotoxicity, because the
normal human stromal cells were unable to form colonies from
single cells under these culture conditions (13). Chart 1 illustrates
the reduction in growth rate of the carcinogen-treated cells at
concentrations from 0.5 to 4.0 //g/ml. Throughout the 16-day
observation period following the first carcinogen treatment, many
vacuolated, multinucleated cells and floating cells were observed.
Solvent-treated control cultures and cultures exposed to a single
0.5-/Kj/ml dose of MNNG did not display a measurable inhibition
diately. Solvent controls received acetone (final concentration 0.5%).
This amount of acetone was not found to be toxic to stromal cells.
Medium controls received no acetone or carcinogen and served to
evaluate effects of solvent on cultures. Both controls were maintained in
parallel to carcinogen-treated cultures. Following the period of exposure,
the Hanks' balanced salt solution was removed, and medium was
replaced. Cultures were either treated repetitively in the same plate, or
they were subcultured to analyze growth characteristics prior to further
treatment. For subcultured cells, phenotypic alterations were analyzed
following each successive carcinogen treatment and subsequent pas
sage. Cultures were examined routinely by phase microscopy to analyze
alterations in cellular morphology. Growth rate, saturation density, and
absolute plating efficiency were analyzed by methods described previ
ously (13, 40).
Histochemical Techniques. GGT (EC 2.3.2.2.) activity was analyzed
as outlined previously (13). Acid phosphatase (EC 3.1.3.2.), alkaline
phosphatase (EC 3.1.3.1.), and leucine aminopeptidase (EC 3.4.11.1.)
were assayed using coupling agents by established techniques (1).
Steroid responsiveness was assayed by first exposing cultures to me
dium containing 0.1 ^M DES for 1 week and then exposing cultures to
medium containing 0.1 ¿IM
DES or to 0.01 MMDES:0.1 UM progesterone
for an additional week. PAS staining of cultures exposed to hormones
was performed with or without prior incubation for 1 hr with a 1%
diastase solution. Mouse epidermis was used as a positive control for
PAS staining and diastase digestion.
Clonal Growth. Clonogenic potential was analyzed by measuring the
capacity for cell growth from single cells in monolayer culture. A suspen
sion of 500, 1000, or 5000 cells was plated in triplicate 60-mm-insidediameter dishes (Falcon); cells attached in 20 hr and were allowed to
grow for 21 days. Dishes were fixed in methanokacetic acid (9:1 ), stained
with 10% Giemsa (Fisher Scientific Co.), and the number of colonies
containing 16 or more cells was determined microscopically.
Anchorage-independent
growth was evaluated by assessing the ca
pacity for clonal growth in soft agar as described previously (13). Briefly,
a base layer of 0.5% agar in medium was used. Cells (105/ml) in medium
were mixed with agar solution to yield a 0.33% agar overlay which was
pipeted onto the base layer. Colonies were allowed to form at 37°for 4
weeks.
Growth in Suspension. Growth in suspension was analyzed by mon
itoring increases in cell number over a 12-day period in spinner flasks.
Cells (1 x 106) were removed from tissue culture plates, added to 15 ml
of medium, placed in spinner flasks and grown at 37°.At 3-day intervals,
a 1-ml suspension was removed from the flasks and counted. Cellular
aggregation was monitored after 2 days by observing macroscopically for clumps of aggregated cells.
Xenotransplantation into Nude Mice. Stromal cells (1 x 106 to 8 x
106) were injected into s.c. and i.p. locations in both adult and newborn
(<48-hr-old) nude mice. Carcinogen-treated cells, receiving from one to
10 MNNG treatments, and acetone-treated cells were injected. In addi
tion, colonies of MNNG-treated stromal cells growing in soft agar were
isolated, mechanically dispersed into a single-cell suspension, and inoc
ulated. Two strains of NIH nude mice, including T-cell-deficient and Band T-cell-deficient animals, were utilized as hosts for the stromal cells.
The mice were observed for 18 months for tumor development. In an
attempt to reduce the immune response observed, a separate group of
JULY
1983
RESULTS
The effects of MNNG treatment were studied in stromal cell
lines derived from 12 individuals. Charts 1 and 2 illustrate rep
resentative results from one cell line. A single 30-min treatment
of cellular growth. The apparent increased growth with 0.5 ng
MNNG per ml was not reproduced in other experiments. At
concentrations of 8.0 ¿tgMNNG per ml, complete cessation of
growth and degeneration of the cultures were observed.6 After
an additional carcinogen exposure, reduced toxicity in response
to the carcinogen was observed. A second exposure to MNNG
at concentrations ranging from 0.5 to 2.0 ^g/ml produced no
growth inhibition when assayed by plating efficiency (Chart 2),
doubling time, and saturation density (data not shown). However,
some inhibition of cellular growth persisted following the second
exposure to 4.0 ng MNNG per ml; this disappeared after the
third treatment.
Multiple treatments with MNNG resulted in cell populations
with an enhanced growth potential as compared to control
cultures. Plating efficiency of medium controls or solvent-treated
endometrial cells averaged approximately 50% (Chart 2). This
was found also for normal stromal cell lines examined after
several passages (13). In early passages, a somewhat lower
plating efficiency was sometimes observed, as is seen for Treat
ments 1 to 3 in Chart 2; these differences presumably are due
to adaptation to culture conditions. Following successive carcin
ogen treatments, cells treated with 4.0 ^g MNNG per ml dis
played increased plating efficiencies after 3 treatments (67%),
and by 4 treatments, they reached 105% (Chart 2). Cells treated
with lower concentrations of MNNG did not show elevations in
plating efficiencies until after the fourth exposure. All the carcin
ogen-treated populations eventually displayed plating efficiencies
of 100%; in different experiments, considerable variation was
observed, but the plating efficiency was always elevated over
that of controls. Cell division of carcinogen-treated cells during
the 20-hr assay may have contributed to these variations. An
increase in plating efficiency (98%) was also observed in another
cell line following a single 1.0-Mg/ml dose of MNNG and subculturing for 56 weeks.
Cells exposed to multiple treatments with MNNG also dis
played increases in saturation density over that of solvent-treated
controls. Saturation density was defined as the number of cells/
sq cm present in multiwell dishes after no further increase in cell
number was demonstrable. The saturation density of control
cells consistently averaged 3.0 x 104 cells/sq cm and showed
6 B. H. Dormán and D. G. Kaufman, unpublished observations.
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S. H. Dormánet al.
6 7 8 9 10 II 12 13 14 15 16
DtMS
Chart 1. Plating efficiency and growth rate of human endometrii cells following
a single treatment with acetone (• •)or with 0.5 (O), 1.0 (x), 2.0 (• •),
and 4.0 (O) t¡gMNNG per ml. Cell cultures were treated once with either MNNG or
acetone and grown for 6 days. Cultures were then removed with trypsin, and the
cell suspensions were plated at a density of 2 x 104 cells/well in multiwell dishes
(10-mm inside diameter). One day following plating and at 4-day intervals thereafter,
cells from duplicate wells were harvested and counted in a Coulter particle counter.
Medium was changed every 4 days.
345678
IO
NUMBER OF TREATMENTS
Chart 2. Plating efficiency of human endometrial cells following repetitive carcin
ogen treatments and subculturing. Cell cultures were treated a total of 10 times
with acetone (• •)
or with 0.5 (O), 1.0 (x), 2.0 (• •),and 4.0 (G) fig MNNG
per ml and were passaged after each carcinogen exposure. Following each
carcinogen exposure and subculturing, cells from each treatment group were
plated in duplicate at densities of 1.2 x 104/sq cm. The number of cells which
attached to the plastic substrata was determined 20 hr after plating; plating
efficiency was defined as the percentage of seeded cells which attached.
little variation over time. In contrast, a 2-fold increase in satura
tion density was observed in carcinogen-treated cultures after 2
to 5 exposures, depending on the amount of MNNG used. The
saturation density of cultures treated with the highest concentra
tion, 4 fig/ml, rose to 7.0 x 10" cells/sq cm after 2 treatments.
At lower MNNG concentrations, similar changes occurred over
a longer period of exposure. These changes correlated well with
morphological observations of cellular crowding.
Cells treated repetitively with MNNG also displayed higher
3350
growth rates than did controls. A progressive decrease in dou
bling time was observed after 3 to 7 exposures, again depending
on the MNNG concentration. Cells treated with 2 or 4 fig MNNG
per ml displayed a slight reduction in doubling time following the
third treatment, and by the seventh exposure, the doubling time
was reduced to 4 days, approximately half that of the controls.
Additional carcinogen treatments (8 to 10) did not further influ
ence the rate of growth. Exposure to 0.5 or 1.0 ¿¿g
MNNG per
ml resulted in similar changes beginning at the fourth treatment.
Doubling time was reduced to 50% of controls by the eighth
treatment. Solvent-treated control populations doubled at an
average of every 7.5 days. Cells treated once with 1.0 ng MNNG
per ml and passaged repetitively over 56 weeks also displayed
a reduction in doubling time to 4 days.
Cells treated with carcinogen have an enhanced life span in
vitro. Over the 2 years of observation, none of the carcinogentreated stromal cells showed signs of senescence. In contrast,
untreated stromal cells lost proliferative capacity after 16 to 18
passages (8 to 10 months), at which time large multinucleated
cells appeared. Eventually, sloughing of late-passage stromal
cells occurred, indicating that normally they demonstrate limited
life span.
Anchorage-independent growth was also examined sequen
tially after each carcinogen treatment. At each of the 4 carcino
gen concentrations used, 4 exposures to MNNG resulted in cells
with the capacity to grow in soft agar as macroscopically visible
colonies from single cells (Fig. 1ß).The colony-forming efficiency
of carcinogen-treated cells in soft agar ranged from 0.018 to
0.069% for different cell lines. Although, in general, the percent
age of cells capable of anchorage-independent growth increased
from the fourth to sixth carcinogen treatment, a clear doseresponse was not seen. After the sixth treatment, the highest
colony-forming efficiency observed in soft agar was 0.069%.
Under the conditions reported here for growth of carcinogentreated cells into colonies in soft agar, medium or solvent-treated
controls were not able to form colonies in soft agar and remained
as single cells (Fig. 1^).
Cultures treated repetitively with MNNG displayed higher lev
els of GGT than did control cells. At all 4 carcinogen concentra
tions, the proportion of treated cells with elevated GGT levels
increased up to the sixth treatment, when most cells displayed
elevated enzyme levels. The most intensely positive cells were
observed to reside in crowded, morphologically altered foci,
where 100% of the atypical, partially overlapping cells stained.
Control populations displayed only faint background levels of
GGT. Two other marker enzymes for endometrial stromal cells
were also demonstrated in carcinogen- and solvent-treated cul
tures. High levels of acid phosphatase and leucine aminopeptidase were demonstrated in control and MNNG-treated cells.
Virtually 100% of the cells displayed histochemically detectable
enzyme activity. In contrast, both normal and carcinogen-treated
cultures were negative for alkaline phosphatase, a marker en
zyme for glandular epithelium in the endometrium.
Steroid responsiveness was tested in a long-term control
culture, in a culture treated 10 times with MNNG, and in cells
derived from a human endometrial stromal sarcoma. Cells ex
posed to DES alone for 2 weeks did not produce substantial
glycogen; there was little material present which was PAS posi
tive and diastase sensitive (Fig. 2, A, D, and G). Minimal cellular
aggregation was observed with DES alone in control and MNNG-
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VOL. 43
Effects of MNNG on Human Endometrial Cells
treated cultures. Some aggregation was always noted in cultures
of the tumor, regardless of steroid treatment. In contrast, cultures
primed with DES for 1 week and then exposed to combined
DES and progesterone displayed marked glycogen production
and cellular aggregation (Fig. 2, B, E, and H). This PAS-staining
material was sensitive to diastase digestion (Fig. 2, C, F, and /).
Solvent-treated cells, cells exposed to MNNG, and the human
tumor responded in this manner.
Carcinogen-treated cultures displayed a progressively atypical
morphology with an increasing number of carcinogen treatments.
Control cells treated with solvent were evenly spaced and uni
form in size (Fig. 3A). The cells had clearly delineated outlines
and regular nuclear size and shape. Nucleoli were small, round,
and predominantly multiple. Nuclearcytoplasmic ratio was low.
A minor degree of cellular crowding in isolated foci transiently
occurred in confluent plates and represented the most severe
morphological alteration observed in control cells (Fig. 36). Fig.
3C illustrates these cells 10 days after a single 1.0-Mg/ml treat
ment with MNNG. In contrast to controls, the cells of the monolayer had a less uniform size and shape. There were areas
displaying cellular crowding and less distinct individual cell
boundaries than normal. Following 2 exposures to MNNG (1.0
i/g/ml) and 4 passages, the cell cultures acquired increasingly
atypical cellular morphology (Fig. 3D). Irregular, small, and
crowded cells represented the major fraction of the cell popula
tion. In areas, cellular crowding progressed to the extent of
blurring cellular outlines, providing an appearance of overlapping
between adjacent cells. Nuclei appeared irregular in size and
shape, and many had single prominent, centrally located nucleoli.
The nucleancytoplasmic ratio also was increased.
After 4 exposures to MNNG, cellular crowding characterized
the entire field (Fig. 3E). There were demarcations between
groups of cells, but individual cells were not clearly evident. The
atypical cellular organization was matched by irregularities in
nuclear size and shape. Many of the cells had single large
nucleoli. Cells treated 6 times with MNNG acquired even more
abnormal morphological characteristics. Fig. 3F illustrates the
edge of a densely crowded focus of cells. Within the focus, the
cells were so intensely crowded that cellular boundaries and
cytological details are obscured. These alterations clearly illus
trate piling up of cells. Some of these foci were of sufficient size
to be visible macroscopically.
The capacity for clonal growth from individual cells was ana
lyzed for carcinogen-treated and control cells. Untreated or sol
vent-treated control cells were not able to grow in a clonal fashion
in CMRL Medium 1066 and degenerated rapidly following lowdensity plating. Cells exposed to one to 5 treatments with MNNG
were also not capable of clonal growth. In cultures exposed to
6 treatments with 1.0 ^g MNNG per ml, cells capable of clonal
growth were detected, with a colony-forming efficiency of 10%.
The stremai sarcoma was also capable of forming colonies in
CMRL Medium 1066 at an efficiency of 3.0%.
Cell growth in suspension and the degree of cellular aggrega
tion were evaluated in a spinner flask for solvent-treated control
cells and for cells treated 10 times with MNNG. Control cell
populations degenerated rapidly in suspension, and after 4 days,
the cultures contained cellular debris and a few remaining single
cells. In contrast, cells treated 10 times with 2.0 pg MNNG per
ml could proliferate in suspension with a generation time of 8
days. These carcinogen-treated stromal cells were also observed
JULY 1983
to aggregate into large clumps 1 to 2 mm in diameter after 2
days.
Carcinogen-treated cells were inoculated into nude mice to
evaluate their potential for tumorous growth. None of the cells
produced tumors in any of the animals during observation pe
riods in vivo of up to 18 months. Nodules that formed at the
injection site 1 to 4 weeks following cell inoculation in several
animals were composed only of polymorphonuclear leukocytes
and macrophages. Stromal cells were not observed in the nod
ules. Histological sections have been taken of injection sites 48
hr following cell inoculation and again revealed immune cells
without evidence of stromal cells.
DISCUSSION
As compared to normal human endometrial stromal cell cul
tures, endometrial cells repetitively treated with MNNG progres
sively demonstrated phenotypic changes which have been as
sociated with cancer: increased growth rate; elevated plating
efficiency and saturation
density; anchorage-independent
growth; clonal growth; detectable and increased levels of GGT
activity; and the ability to proliferate in suspension. The cells also
acquired an increasingly abnormal morphology after carcinogen
exposure. The effectiveness of the carcinogen to produce these
changes was related to its ability to produce cytotoxicity. There
was a tendency for cells treated with the higher carcinogen levels
to acquire the increased growth capacity earlier than those
exposed to lower MNNG concentrations. Thus, it appears that
the cumulative exposure to carcinogen is related to the appear
ance of increased growth potential. Solvent-treated cells did not
display any of these alterations, even after multiple solvent
treatments and subculturings. In addition, control cells under
went senescence after more than 18 passages, while carcino
gen-treated cells have been continuously cultured for over 2
years. This suggests that the alterations are a direct result of
the carcinogen treatment and are a progressive change, rather
than a selection of cells with increased growth potential within
the original population. A single carcinogen treatment is also
effective in inducing elevated growth capacity, although many
months of subculturing were required before the cells expressed
any alteration in phenotype. Since cells treated repetitively with
carcinogen acquired an enhanced growth capacity much earlier,
repetitive treatments appear to accelerate the expression of
altered phenotypes, even though the cells become resistant to
the toxic effects of MNNG.
Each of the observed phenotypic changes has been associ
ated with transformation in vitro. Increases in plating efficiency,
growth rate, and saturation density have been observed in many
mammalian cell lines following carcinogen treatment (5, 22, 31).
Several investigators have shown that increases in saturation
density and growth rate were necessary prerequisites but not
sufficient criteria for cancer in a variety of mammalian cell lines
(15,19, 24). The acquisition of the capacity for clonal growth, in
systems in which cells were incapable of cloning before carcin
ogen treatment, has been shown to be a property of cells in the
transition to cancer (39).
Elevated levels of GGT have been demonstrated in epithelial
cells from laboratory animals chemically transformed in vitro, in
preneoplastic lesions of rat liver, and in some human tumors (18,
34). This enzyme may serve as a useful marker in determining
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B. H. Dormánet al.
the acquisition of atypical biological states, especially since we
found the most intensely positive cells residing in foci containing
highly atypical cells.
The propensity for cellular aggregation, which we observed in
carcinogen-treated cells grown in suspension, has been corre
lated recently with a malignant phenotype in chemically treated
cells (30). Morphological alterations such as altered nuclearcytoplasmic ratios, irregular cell and nuclear shape, and
piling up of cells have been shown to be properties of both
transformed cells and cells that are progressing towards trans
formation in vitro (2, 8, 34). Such changes observed in human
stromal cells are consistent with cellular changes characteristic
of a neoplastic phenotype. In fact, when examined by a cytopathologist, the severely altered MNNG-treated cells have been
interpreted as malignant.
Finally, anchorage-independent
growth is perhaps the most
commonly used index of transformation; the marker is highly
correlated with tumorigenicity (3, 9, 11, 14). The colony-forming
efficiency of stromal cells in agar, 0.018 to 0.069%, was lower
than the efficiencies observed with cells from experimental ani
mals (5 to 9%) and did not continue to increase after 6 carcinogen
treatments. Colony-forming efficiency in agar is subject to culture
conditions, and the observed values may not represent an opti
mal efficiency in agar. Even though a small number of cells
expressed this phenotype, it still represents a clear transition in
comparison to control cells and in comparison to cells exposed
to fewer than 4 MNNG treatments. Stromal sarcoma cells were
also able to form colonies in agar at an efficiency of 0.02%.7
Despite the deviations from normalcy which were documented
in the carcinogen-treated cultures, the cells still exhibited several
important properties of stromal cells. Two characteristic en
zymes, acid phosphatase and leucine aminopeptidase (10), were
expressed by normal cells and cells exposed repetitively to
MNNG. Alkaline phosphatase, a marker for endometrial epithe
lium (10), was not expressed by either normal or carcinogentreated cells. In addition, both normal and transformed cultures
produced glycogen, as evidenced by diastase-sensitive material,
following steroid hormone stimulation; this was also true of cells
cultured from a human stromal sarcoma. The sarcoma was also
positive for GGT,7 was able to form colonies from single cells,
and displayed overlapping of cells which was similar to that seen
in MNNG-treated cultures. These observations demonstrate that
the transformed cells are stromal in origin and have several
properties in common with a tumor of stromal cell origin. It is
unlikely that the altered cells represent an abnormal subpopulation which simply has been selected by carcinogen treatment.
Although we have succeeded in altering many properties of
these human cells with MNNG, we have been unable to obtain
tumors following xenotransplantation into nude mice. Neither
normal nor carcinogen-treated stromal cells survived more than
a few days in vivo after we inoculated them into nude mice under
the conditions specified earlier. Since the cells are unable to
become established under conditions we have used, their ability
to form tumors cannot be adequately evaluated. Although a
positive result would be confirming evidence of transformation,
a negative result does not prove the cells are not transformed.
In fact, many human tumors (36) and cell lines derived from
human tumors (16) are unable to grow in nude mice. No results
7J. M. Siegfried, unpublished observation.
3352
are as yet available for experiments in which stromal sarcoma
cells were inoculated in nude mice.
Results obtained with human endometrial stromal cells agree
with previous observations by others on the difficulty of trans
forming human cells. It has been suggested that stability of
ploidy and enhanced repair capabilities contribute to the difficulty
of transforming human cells (29). Despite apparent overall differ
ences in susceptibility to transformation, human cells and cells
from experimental animals undergo similar types of carcinogeninduced alterations. These similarities suggest that the process
of transformation in different species may progress through
similar pathways.
Repetitive treatment of stromal cells with MNNG induces a
series of biological changes which are acquired progressively
through several stages. Although the cellular changes appear
sequential, experimental data to date are insufficient to confirm
this impression. Well-defined temporally distinct steps are ab
sent. A number of the phenotypic alterations are expressed
within this mixed-cell population gradually over several carcino
gen treatments and therefore overlap between stages. Early
events in the process of cellular change primarily involve en
hanced growth potential, characterized by increases in plating
efficiency and saturation density. Cells at this stage also show
limited cellular crowding but do not display any of the other
phenotypic alterations. With additional carcinogen treatment,
cells that have expressed early changes in growth potential
acquire the capacity to grow in soft agar and demonstrate
elevated levels of GGT. Concurrently, pleomorphic cells and
nuclei become evident, and the nuclearcytoplasmic
ratio in
creases. Increases in growth rate also begin during this stage.
Finally, following additional exposure to carcinogen, cells acquire
the capacity for clonal growth in plastic substrata, a late event
which is observed only after the cells have progressed through
other phenotypic alterations. Severe morphological changes and
the formation of piled-up foci also appear relatively late compared
to other cellular alterations. The progressive development of
separable broad classes of phenotypic changes provides evi
dence that carcinogen-induced changes proceed through multi
ple steps in human endometrial stromal cells. This progressive
nature of transformation has also been shown to be a common
feature in the evolution of many human cancers. These findings
suggest that studies of chemical carcinogenesis in human cells
in vitro may eventually offer insights into the process of human
cancer development in vivo.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance of Carol A. Klevay, Patricia W. Bryant,
and Marc J. Mass in preparation of this manuscript.
REFERENCES
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S. H. Dormánet al.
:
Fig. 1. Anchorage-independent growth of human endometrial cells. A, acetone-treated control cells subcultured over a 14-week interval; B, cells treated 6 times with
MNNG (1 0 »jg/ml)and passaged 8 times over a 14-week interval. Cells (1 x 10s) (in 0.33% agar) were plated onto a 0.5% agar base layer. Four weeks following plating,
the agar plates were examined for macroscopically visible colonies; phase microscopy, x 400.
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VOL. 43
Effects of MNNG on Human Endometrial Cells
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Fig. 2. Steroid responsiveness of cultured endometrial stromal cells. A, acetone-treated control cells exposed to DES alone; B, control cells exposed to
DES:progesterone; C, diastase digestion of B; D, carcinogen-treated cells (1 ^9 MNNG per ml, 10 times) exposed to DES alone; E. carcinogen-treated cells exposed to
DES:progesterone; F, diastase digestion of £;G, stromal cell sarcoma exposed to DES alone; H, stromal cell sarcoma exposed to DES:progesterone; /, diastase digestion
of H. Cells were seeded on chamber slides at a density of 1.5 x 10* cells/sq cm and subjected to steroid treatment as described in "Methods." Slides were fixed in 10%
formalin: 90% ethanol and stained prior to light microscopy. PAS, x 200.
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3355
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VOL. 43
Effects of MNNG on Human Endometrial Cells
ra*
.
3C
3FFig. 3. Morphological alterations of human endometrial cells following multiple treatments with MNNG and solvent. A, control cell populations treated with acetone 3
times and passaged 6 times over a 7-week interval; B, control cell populations treated with acetone 10 times and passaged 14 times over a 26-week interval; C, cells 10
days after a single exposure to 1.0 /jg MNNG per ml; D, cells treated twice with 1.0 »gMNNG per ml and passaged 4 times over a 6-week interval; E. cells treated 4
times with 1.0 ^g MNNG per ml and passaged 6 times over a 13-week interval; F, cells treated 6 times with 1.0 ^g MNNG per ml and passaged 16 times over a 24week interval; phase microscopy, x 400.
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3357
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Alterations of Human Endometrial Stromal Cells Produced by N
-Methyl-N′-nitro-N-nitrosoguanidine
B. Hugh Dorman, Jill M. Siegfried and David G. Kaufman
Cancer Res 1983;43:3348-3357.
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