Download Microfluorometric Analysis of DNA Content Changes in a Murine

[CANCER RESEARCH
36, 1894-1899, June 1976]
Microfluorometric Analysis of DNA Content Changes in a
Murine Teratocarcinom&
Douglas E. Swartzendruber,2
L. Scott Cram, and John M. Lehman
Agriculture Biosciences Group (D. E. S.Jand Biophysics and Instrumentation Group fL. 5. CI, Los Alamos Scientific Laboratory, University of California, Los
Alamos, New Mexico 87545, and Department of Pathology, University of Colorado Medical Center, Denver, Colorado 80220 (J. M. L.J
SUMMARY
The multipotential stem cell of the munine teratocarci
noma, embryonal carcinoma (EC), is capable of differentia
tion in vivo and in vitro to nonneoplastic progeny. Undiffer
entiated EC cells, spontaneously differentiating teratocarci
noma cells, and differentiated cells derived from EG cells
were analyzed for DNA content and chromosome number
distributions. Flow microfluorometnic and fluorescence cy
tophotometnic analysis of DNA content showed that EG cells
had a characteristic diploid (2c) distribution, whereas sev
eral differentiated cell lines derived from EG cells had 4c
DNA distributions. The tetraploid cell populations studied
were capable of cell division but had restricted differentia
tive potential and were either of low tumonigenicity or non
tumorigenic. In vivo teratocarcinomas, comprised of both
EG cells and differentiated cell types, contained diploid and
tetraploid populations. Ghromosomally, EG cells were near
diploid (39 chromosomes) and differentiated cells were
near-tetraploid (62 to 76 chromosomes). The teratocarci
noma provides a model for studying the basic mechanisms
that control the growth dynamics of the rapidly and slowly
proliferating cell populations present in many tumors.
INTRODUCTION
Munine testicular teratocarcinomas are comprised of ma
lignant totipotential stem cells, termed EC,' as well as dif
ferentiated tissues representative of the 3 embryonic germ
layers (24, 33). The EC cells are responsible for the malig
nancy of these tumors (16), whereas the differentiated prog
eny of EC cells are generally benign (25). The EG cells have
characteristics similar to those of cells in the early mouse
embryo (20, 23) which include multipotentiality and ultra
structural, biochemical, and surface properties. The undif
ferentiated (totipotential) stem cells and their differentiated
(unipotential) progeny can be grown in tissue culture (20,
21 , 23), and morphological, biological, and biochemical
parameters of in vitro differentiation of EG cells have been
described (20, 23).
The teratocarcinoma provides an attractive model to
1 This
work
was
supported
by
Grants
CA-16030,
CA-13419,
and
CA-i
5823
from the National Cancer Institute, by a grant from the Milheim Foundation,
and by the United States Energy Research and Development Administration.
a To whom
3 The
requests
abbreviations
for reprints
used
are:
EC,
should
embryonal
be addressed.
carcinoma;
FMF,
flow
micro
fluorometry; FC, fluorescence cytophotometry; PYS. parietal yolk sac; SE,
squamous epithelium.
Received December ii , 1975; accepted February 13. 1976.
1894
study the conversion of malignant cells to benign cells in
cancer. That less differentiated malignant cells can give rise
to differentiated benign cells has been observed in primary
tumors (e.g. , conversion of a neuroblastoma to a gangli
oneuroma) and has been documented in several expeni
mental tumor systems, both in plants and in animals (3-5, 8,
24-26, 38). In vitro maturation of tumor cells has been
demonstrated in a variety of mouse tumors (3, 9, 18, 21 , 2729).
Interestingly, the karyotypes of cells from munine testicu
lar teratocarcinomas, early in their development, are similar
to those of normal testicular cells (32). Unlike most cell lines
derived from mouse tumors, nearly all lines of EG cells
reported have diploid or near-diploid karyotypes, whereas
karyotypes of the differentiated derivatives of EG cells range
from apparently normal to highly aneuploid (20, 23). FMF
(13, 36) and FC were used in this study as methods for rapid
and reproducible quantitation of the DNA content of undif
ferentiated and differentiated teratocarcinoma cells. The EC
cells have characteristic diploid DNA and chromosome dis
tributions, and lines of differentiated cells that we have
obtained from cultures of EG cells have tetraploid DNA and
chromosome distributions. Kinetic analysis of sponta
neously differentiating EG cells showed that, as differen
tiated cells appear, the DNA distributions shift progressively
from diploid to tetraploid. The change in DNA content of
teratocarcinoma cells is correlated to the morphological,
biological, and biochemical differentiation of these cells.
MATERIALS AND METHODS
Tumor. The teratocarcinoma (OTT 6050) was obtained
from Dr. Leroy Stevens. We have maintained the stock
tumor for over 4 years in 129/J mice (Jackson Laboratories,
Bar Harbor, Maine) either s.c. or i.p. (24).
Cells. Isolation of EG cell lines and of differentiated tera
tocarcinoma cells from solid tumors has been described
previously (22, 34). Cell lines were maintained in Dulbecco's
modified Eagle's medium (Grand Island Biological Go.,
Grand Island, N. V.), supplemented with 5% fetal calf serum
and antibiotics (100 IU penicillin per ml and 100 j.tg strepto
mycin per ml), and were subcultured using 0.25% trypsin.
EG cell lines maintained by frequent subcultuning (every 2
days) are nearly 100% stem cells; however, when infre
quently subcultured, differentiated cells appear at the pen
phenies of the EG colonies (22). The differentiated cell lines
used were PVS-1 and PVS-2 (22) and SE-i (D. E. Swartzen
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DNA Content Changes in a Teratocarcinoma
druber, manuscript in preparation). These lines were de
rived from cultures of EC cells. They are morphologically
distinguishable from EC cells, and they no longer contain
any multipotential EC cells (22, 34). When EC cells were
injected s.c. into 129/J mice (106cells/0.25 mI/mouse), 10mm teratocarcinomas formed within 28 days. The PVS cell
lines grew much more slowly in vivo, if at all, and often
required up to 6 months to form palpable nodules. These
tumors were not teratocarcinomas; instead, they were PVS
tumors (22). The SE cell line is nontumorigenic (D. E.
Swartzendruber, manuscript in preparation). Other markers
of differentiation of EC cells were also monitored. These
included changes in cell volume, synthesis of basement
membrane by the PVS and SE cell lines, susceptibility to
SV4O infection of spontaneously differentiating EC lines
and differentiated lines, and histochemical localization of
alkaline phosphatase levels in undifferentiated and differen
tiated teratocarcinoma cells. The techniques for these as
says were the same as previously described (2, 22, 34). The
procedure for cytogenetic analysis of each cell line was also
the same as described (22).
FMF and FC Analysis. Tissue culture cells were treated
with trypsin-EDTA (35) for 5 mm to produce a suspension of
single cells. The cells were stained for 20 mm using the
mithramycin procedure (6) which specifically stains DNA.
Single-cell suspensions were prepared from solid tumors by
forcing tumor tissue through a small-mesh steel screen with
a glass syringe plunger into a culture dish containing phos
phate-buffered saline (Na2HPO4, 1.17 g/liter; NaH2PO4,
0.224 g/liter; NaCI, 8.5 g/liter). Clumps were allowed to
settle, and the supernatant containing >95% single cells
was decanted, spun down, and suspended in mithramycin
stain. Stained cells were individually measured for their
DNA content by FMF analysis. The fluorescence intensity of
the intracellular dye bound to DNA was measured for each
cell (5 x i0@cells/mm) and was recorded in a multichannel
pulse-height analyzer. Details of the instrumentation have
been published elsewhere (13).
For FC analysis, coverslips on which the teratocarcinoma
cells were grown were washed once with 0.9% NaCI solu
tion, and their intracellular DNA was fluorescently stained
using the propidium iodide procedure of Knishan (19). The
DNA content of individual nuclei was then measured using a
Zeiss fluorescence cytophotometer equipped with a digital
readout. The fluorescence intensity (DNA content) of mdi
vidual cells was then correlated to cellular morphology us
ing phase optics.
chemically (22). Inversely, the morphologically d ifferen
tiated cells (in both spontaneously differentiating and pure
cultures) contained low levels of alkaline phosphatase,
were susceptible to infection with SV4O, and were low- or
nontumonigenic. Fig. 1 presents an example of the sponta
neous in vitro differentiation of EC cells. At 24 hr after
subcultuning, colonies consist of nearly 100% EC cells (Fig.
1A). After 3 days, spontaneous differentiation is seen at the
periphery of the colony of EC cells (Fig. 1B), and by Day 10
several cell types, including epithelium, spindle, and neu
ron-like cells, can be recognized (Fig. 1C). Fig. 1D shows a
culture consisting only of differentiated PVS cells. In con
trast to EC cells, these cells produce large amounts of
basement membrane. Kinetic analysis of the Coulter volume
of cells in cultures of spontaneously differentiating EC cells
showed that, as differentiation progressed, populations of
cells with volumes greater than the EC cells appeared. PYS
cells have an average volume twice that of EC cells.
Chromosome Analysis. The chromosome number distri
butions of EC cells, spontaneously differentiating teratocar
cinoma cells, and differentiated PVS cells are shown in
Chart 1. The EC cells, when initially established in vitro (22),
had a stem line chromosome number of 39, and this has
remained the same after 3 years in tissue culture (Chart 1A).
As differentiated cells appear in the cultures, cells in the
near-tetraploid range appear (Chart 1B). The 3 lines of
differentiated cells analyzed (PVS-1, PVS-2, and SE-i ) each
‘I)
L)
0
z
RESULTS
Criteria for in Vitro Differentiation of Teratocarcinoma
Cells. Previous work has established several parameters
pertinent to teratocarcinoma differentiation (2, 20, 22-24,
34). Several of these differentiation-related markers were
monitored during these experiments and, in agreement with
previous results, the morphologically undifferentiated EC
cells retained their multipotentiality in vitro and in vivo and
were highly tumonigenic. They contained high levels of al
kaline phosphatase when measured histochemically (2),
they resisted infection with SV4O (34), and they produced
little basement membrane when measured immunohisto
NUMBEROF CHROMOSOMES
Chart 1. Chromosome distribution histograms of 3 lines of teratocarci
noma cells. A, undifferentiated stem cells; B, spontaneously differentiating
EC cells (Day 15); C. differentiated derivative of EC cells (PYS-1). More than
50 mitoses in each group were examined.
JUNE 1976
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1895
D. E. Swartzendruber
et al.
had chromosome number distributions in the near-tetra
ploid range (62 to 76 chromosomes; Chart 1C). There was
no evidence of gross chromosomal abnormalities (breaks or
dicentnic, ring, or metacentnic markers) in any of the lines
analyzed.
FMF Analysis. Since changes in chromosome number do
not necessarily correlate to equivalent changes in DNA con
tent (17), the relative DNA content of various teratocarci
noma cells was measured. FMF was used to measure the
DNA content of EC cells, EC cells and differentiated cells in
spontaneously differentiating cell cultures, differentiated
teratocarcinoma cells, and cells from tumors induced by
each of these cell preparations. Chart 2 shows the DNA
distributions of several cell populations, and Table 1 gives
the percentages of cells (area) in each of the cell cycle
compartments. Munine spleen cells were used as a control
for determining the values for normal 2c (G,) and 4c (G2 +
M) amounts of DNA. When cultures of EC cells were ana
lyzed for DNA content 24 hr after subcultuning, the great
majority of the cells were diploid (i.e., their G, peak was in
the same channel as that of normal spleen cells) (Chart 2;
Table 1). In spontaneously differentiating cultures at 5, 10,
and 40 days after subcultuning, FMF analysis showed an
increase in the percentage of cells with a tetraploid amount
of DNA and a concomitant decrease in the percentage of
cells with diploid DNA (Chart 2, C and D; Table 1). As the
differentiated cells reached confluency (Day 40), the num
ben of tetraploid cells synthesizing DNA and undergoing
mitosis decreased to <15% (Table 1).
When cultures of differentiated teratocarcinoma cells
(PVS-1, PVS-2, SE-i) were analyzed for DNA content, it was
shown that all cells in each line had a tetraploid DNA distni
bution (Chart 2E; Table 1). Moreover, when cells from dip
bid or tetraploid cell lines were injected back into the
animals, diploid lines (EC cells) produced teratocarcinomas
containing populations of diploid and tetraploid cells (Chart
2F; Table 1), whereas the tetraploid PVS-i cell line pro
duced tumors in which the great majority of cells had a
tetraploid DNA distribution (Table 1). Diploid cells present
in the tetraploid tumors presumably were of host origin
since, when these tumors were placed in tissue culture, the
diploid population decreased rapidly (Table i).
FC Analysis. Microscopic cytofluorometnic measure
,
-_-1I@I@_
cells
Teratoccwcanomo
in vitro
I
I
Differentioted___,@
Normal spleen
‘
5
@
Teratocycinoma
in
Day I
.00
5
0
5.
C
A
Teratocarcinoma
in vitro
@
I
Day 3
@EmbryonaI•@,
carcinoma
@itro
Differentiated________
cells
cells
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‘0
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,‘
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in vitroTeratococinoma
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.0
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Differentiated teratocarcir@‘a
cells (PYS) in vitro
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(PYS)
in
Terotocorcinoma in vivo
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IC
)J\@EJ@@:
0
20
40 60 80 100 0
20 40 60
ChannelNumber (DNA content)
80
00
Chart 2. FMF analysis of DNA content and cell cycle distribution of tera
tocarcinoma cells. A, normal spleen cells; B, EC cells 1 day after subcultur
ing; C, EC cells 5 days after subculturing; D, EC cells 10 days after subcultur
ing; E, differentiated teratocarcinoma cells (PYS); F, teratocarcinomain vivo.
The percentages of cells in each compartment of the cell cycle are listed in
Table 1.
i 896
(-@ C
-
0
I
5
0
5
Fluorescence (DNA content)
Chart 3. FC analysis of the DNA content of single teratocarcinoma cells
in culture. A, EC cells 1 day after subculturing; B, EC cells 3 days after
subculturing;
C, differentiated
teratocarcinoma
cells (PYS). The data on the
DNA content of undifferentiated and differentiated teratocarcinoma cells can
be correlated
to the morphological
analysis in Fig. 1.
CANCER RESEARCH VOL. 36
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DNA Content Changes in a Teratocarcinoma
Table 1
FMFanalysisof DNA content of teratocarcinomacells
% of
in4DiploidG,
cells
+ M
tetra
G,Cell
MNormal
typeGSG,―ploid
Tetra
ploid
DiploidDiploidploidTetra
S+
(control)934300ECin
spleen
vitro , Day 148311
821in
77in
vitro, Day 5351
vitro, Day 101
8302413in
32Differentiated
vitro, Day 401
5261
51
51
2581
teratocarci
cellsSE-I
noma
vitro)00502624PYS-l
(in
vitro)00283735PYS-Il
(in
vitro)00322741ECinvivo551217133in
(in
13Differentiated
vivo -@in vitro,
48 hr4320231
teratocarci
cellsPYS-l
noma
vitro)00235225PYS-I
(in
(in vivo)1
619PYS-I
vitro,1133293648
(in vivo —v
in
91451
hr)
a Data
from
the percentage
FMF-generated
distribution
curves.
Area
under
the
curve
is proportional
to
of cells in each phase of the cell cycle (7); >10@ cells in the peak channel;
data representativeof at least 4 replicate experiments.
b Data
given
as
1 percentage
since
the
DNA
content
of
diploid
G,
+
M
cells
is equivalent
tothatoftetraploid
G cells.
ments were made of the DNA content of individual cells in
cultures of undifferentiated, spontaneously differentiating,
and differentiated teratocarcinoma cells. The DNA content
of single cells was then correlated with cellular morphology
by direct visualization of each cell using phase optics. At 24
hr after subculturing (Fig. iA), the cell colony consisted
almost entirely of EC cells, although a few morphologically
differentiated cells were present. As shown in Chart 3A, the
earliest differentiated cells identifiable at the colony peniph
ery at 24 hr contained amounts of DNA in the tetraploid
range, even though the great majority of cells in the culture
were diploid (as shown by FMF in Chart 2B). The tetraploid
population detectable by FC increased with progression of
spontaneous differentiation (Chart 3B) and parallels the
increase in this population as demonstrated by FMF (Chart
2, C and D). FC analysis of differentiated PVS cells showed
that all these cells had a tetraploid DNA distribution (Chart
3C).
DISCUSSION
Using cytogenetic analysis, FMF, and FC, we have shown
that, in our teratocarcinoma cell lines, the appearance of
differentiated cells in cultures of stem cells is correlated
with an increase in chromosome number, as well as with a
doubling of DNA content from diploid to tetraploid. As has
been shown previously, changes in chromosome number
are not necessarily correlated with changes in DNA content
(17). Our data show that, both in vivo and in vitro, EC cells
contain diploid DNA distributions. The earliest morphologi
cal differentiation of EC cells in vitro is apparently associ
ated with a doubling of the DNA complement. Also, progeny
of EC cells that are greatly limited in both their differentia
tive and proliferative capacities have tetraploid DNA distni
butions.
Although changes in chromosome number and DNA con
tent are found in a variety of tumors, it is not known what
relationship these shifts have to differentiation in the terato
carcinoma system. It is possible that the majority of differ
entiated cells are arrested in diploid G2 (tetraploid G,) and
that cells contributing to the proliferating tetraploid popula
tion are either specific types of differentiated cells or spon
taneously transformed differentiated cells. Another possi
bility
is that
an increase
in chromosome
number
and
DNA
content from diploid to tetraploid may be a necessary pre
requisite for the differentiation of EC cells. That diffenentia
tion in this subline of the teratocarcinoma is apparently
accompanied by a doubling of the DNA content does not
alter the fact that this tumor mimics many aspects of normal
differentiation. Induced tetraploidy in mouse eggs does not
prevent normal development of the embryos to term (31).
The spontaneous appearance of tetraploid cells in the tera
tocarcinoma system may provide a means for monitoring
differentiation in this tumor. Also, alterations of DNA con
tent may be important in growth regulation of this tumor.
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1897
D. E. Swartzendruber et al.
ii . Hitosumachi, 5., Rabinowitz, z., and Sachs, L. Chromosomal Control of
Our preliminary resultsindicate that EC cells may be stimu
latedto differentiate
more rapidly
by inducingtetraploidy Reversion in Transformed Cells. Nature, 231: 511-514, 1971.
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with Colcemid, cytochalasmn B, or bromouracil deoxynibo
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The correlation between DNA content changes and differ
14. Horan, P. K., Jett, J. H., Romero, A., and Lehman, J. M. Flow Microfluo
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@
@.d
.
DNA Content Changes in a Teratocarcinoma
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Fig. 1. Phase micrographs of spontaneously differentiating EC cells. A, EC cells 1 day after subculturing [little morphological differentiation (DIFF) can be
seen at the periphery of the piled-up cells]; B, EC cells 3 days after subculturing (differentiated cells are readily visualized at the periphery of the EC cell
colony); C, EC cells 10 days after subculturing (EC cells have given rise to numerous differentiated cell types); D, culture of a differentiated line
teratocarcinoma cells (PYS) (no EC cells are present).
JUNE 1976
Downloaded from cancerres.aacrjournals.org on August 1, 2017. © 1976 American Association for Cancer Research.
1899
Microfluorometric Analysis of DNA Content Changes in a
Murine Teratocarcinoma
Douglas E. Swartzendruber, L. Scott Cram and John M. Lehman
Cancer Res 1976;36:1894-1899.
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Downloaded from cancerres.aacrjournals.org on August 1, 2017. © 1976 American Association for Cancer Research.