[CANCER RESEARCH 42. 4999-5003,
0008-5472/82/0042-OOOOS02.00
December 1982]
Kinetic Heterogeneity in Density-separated
Subpopulations1
William A. Brock,2 Douglas E. Swartzendruber,
Murine Fibrosarcoma
and David J. Ordina
Department of Experimental Radiotherapy, University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 [W. A. B., D. J. GJ,
and Department of Biology, University of Colorado at Colorado Springs, Colorado Springs, Colorado 80933 [D. E. SJ
ABSTRACT
Murine fibrosarcoma cells can be separated into subpopu
lations by centrifugation through 10 to 35% Renografin density
gradients. Previous work has shown that the heavier cell pop
ulations are rich in chronically hypoxic cells. In this study, each
subpopulation was characterized for thymidine incorporation,
thymidine transport, thymidine triphosphate pool sizes, and
thymidine triphosphate specific activities. The heavier cell pop
ulations have less accessibility to exogenous thymidine, and
they have lower endogenous pools of thymidine triphosphate
and synthesize lower levels of DNA than do the lighter cell
populations. However, if the cells are removed from the tumors
and labeled with [3H]thymidine in vitro, all subpopulations syn
thesize DNA at similar rates. Two-parameter flow cytometry
using acridine orange staining following partial acid denaturation of chromatin identified a small quiescent population in the
most dense cell fraction, but the small number of these cells
cannot account for the results of the biochemical studies. It
appears that the hypoxic cells in the fibrosarcoma tumors are
noncycling or slowly cycling, are in all phases of the cell cycle,
and recover their ability to synthesize DNA when cultured
under in vitro conditions.
INTRODUCTION
Cells isolated from a murine FSA3 can be separated into 5
distinct subpopulations by centrifugation through Renografin
density gradients. These subpopulations differ in clonogenic
ability, as shown by lung colony formation (7), and in their
sensitivity to both low- (5) and high-linear energy transfer
radiation (6). The radiation response suggests that the moredense cell populations contain a high fraction of hypoxic cells.
This conclusion is supported by studies of FSA tumors irradi
ated in mice made acutely hypoxic (4) and in mice treated with
hypoxic cell sensitizers (8).
It has been reported recently that the LI of the relatively lowdensity cells is the same as the percentage of S-phase cells in
these fractions, while the LI of the denser cells is only about
one-half of that predicted by the percentage of S-phase cells
(9). This suggests that the S-phase cells in the more dense
populations are stationary, synthesize DNA very slowly, or
receive inadequate labeled precursor. All possibilities are con1 This work was supported in part by Research Grants CA-18628, CA-06294,
and CA-16672, awarded by the National Cancer Institute.
* To whom requests for reprints should be addressed, at Department of
Experimental Radiotherapy. M. D. Anderson Hospital and Tumor Institute, 6723
Bertner Avenue, Houston, Texas 77030.
3 The abbreviations used are: FSA, fibrosarcoma; LI, labeling index; FCM. flow
cytometry; AO. acridine orange: dsDNA, double-stranded DNA; ssDNA, singlestranded DNA.
Received May 18, 1982: accepted September 8, 1982.
DECEMBER
1982
sistent with a cell population that is isolated from the functional
vasculature of the tumor.
In this study, we examined DNA metabolism in isolated FSA
subpopulations in greater detail by measuring in vivo and in
vitro [3H]thymidine incorporation and LI, TTP specific activities
and pool sizes, [3H]thymidine uptake, and 2-parameter FCM
using AO. Our purpose was to characterize further, in terms of
tumor biology, the variant DNA metabolism exhibited by the
more-dense cell populations.
MATERIALS
AND METHODS
Materials. The [5-metfjy/-3H]thymidine
(specific activity, 50 Ci/
mmol), [5-me</7y/-3H]TTP (specific activity, 42 Ci/mmol), and [MC]dCTP
(specific activity, 452 mCi/mmol) were obtained from Amersham/
Searle Corp. (Arlington Heights, III.). All nonradioactive deoxyribonucleoside triphosphates (TTP, dCTP, dATP, and dGTP) were obtained
from Sigma Chemical Co. (St. Louis, Mo.). Escherichia coli DNA
polymerase I and nicked calf thymus DNA were purchased from Bethesda Research Laboratories (Gaithersburg, Md.). Renografin-60 is a
product of Squibb and Sons (Princeton, N.J.). AO was purchased from
Polysciences, Inc. (Warrington, Pa.). The balanced salt solution con
tained 8 g NaCI, 0.4 g KCI. 1.0 g glucose, and 0.35 g NaHCO3 per liter
water.
Animals. Eight- to 10-week-old C3Hf/Kam mice were used for tumor
growth. These mice were syngeneic with the FSA.
Tumors. The FSA used in these experiments was induced with
methylcholanthrene
(11), and, after 5 generations of isotransplants,
was stored frozen; thus, experiments, both present and published
previously, used a comparable tumor. Experimental tumors were
formed by the s.c. injection of 3 x 10s cells into the hind legs of the
animals, and the tumors were used after they reached a 12-mm
diameter. Single-cell suspensions were prepared by a protocol using
trypsin and DNase I published previously (7).
Density Gradient Centrifugation.
Suspensions
containing
5 x 10r
tumor cells in 3.0 ml of culture medium were layered over 34-ml 10 to
35% linear gradients of Renografin-60 diluted with Ringer's solution
and containing DNase I (200 units/ml) (type I; Sigma Chemical Co.)
(7). The gradients were centrifuged at 13,000 x g at 4°for 30 min,
and 3 ml were removed from each band of cells by aspiration through
a 25-gauge needle. The cells were finally washed twice with a balanced
salt solution to remove the Renografin.
Thymidine Labeling. Mice bearing 12-mm tumors were given injec
tions of 100 fiCi of [mef/7y/-3HJthymidine (specific activity, 53 Ci/mmol;
Schwarz/Mann,
Spring Valley, N. Y.) and sacrificed 1 hr later. The
tumors were removed, and a single-cell suspension was prepared as
described above. For in vitro labeling, tumor cells were first suspended
in McCoy's modified Medium 5A with 5% calf serum at 5.0 x 107
cells/ml. Next, [3H]thymidine (6 Ci/mmol) was added for a final con
centration of 0.1 /iCi/ml, and the mixture was incubated for 15 min in
a shaking water bath at 37°. After incubation, the cells were cooled
rapidly on ice, and 3-ml aliquots of the suspension
Renografin gradients.
TTP Pools and Specific Activities.
density populations were determined
were layered over
The TTP levels in the different
by the method of Solter and
4999
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W. A. Brock et al.
Handschumacher (10) with some modifications. In each experiment,
FSA cells were density banded on 6 density gradients, and the corre
sponding fractions were pooled. Between 8 x 107 and 2 x 10e cells
from each density fraction were extracted with 2.0 ml of 0.5 M HCIO4
at 4° for 2 hr, followed by a second extraction with 1.0 ml. The
combined extracts were neutralized to pH 7.4 with KOH, centrifuged
to remove the KCIO4 crystals, and lyophilized. The lyophilized powder
was resuspended in 0.5 ml of distilled water and centrifuged to remove
undissolved crystals of KCIO4, and samples of up to 0.1 ml were used
for each assay. The reaction mixture contained [MC]dCTP adjusted to
contain 150 ng dCTP and 20,000 cpm, 300 ng dGTP, 300 ng dATP,
and 7 /ig nicked calf thymus DNA in a total volume of 0.2 ml reaction
buffer [90 mw Tris-HCI (pH 7.4): 4 mM MgCI2:1.3 mw 2-mercaptoethanol]. The reaction was started by adding 0.1 ml of E. coli DNA
polymerase I (3.5 units). Standards contained between 10 and 100 ng
"FTP. After 60 min, the samples were cooled on ice, 0.3 ml of cold 33%
trichloroacetic acid were added to each tube, and the suspension was
transferred to a GF/C filter. The filters were washed with 10 ml of cold
5% trichloroacetic acid and then dried after being washed with 5 ml of
95% cold ethanol. The radioactive content of each filter was then
determined by liquid scintillation counting.
To determine the specific activity of the intracellular TTP pools, cells
were first labeled with high-specific-activity
[5-mei/?y/-3H]thymidine and
then extracted and assayed as described above. In a separate exper
iment, the fraction of total [3H]TTP incorporated into the calf thymus
DNA by polymerase I was determined in the presence of several
different concentrations of nonradioactive TTP. Therefore, by using
double-label scintillation counting, the total amount of TTP can be
determined from the MC content, and the fraction of [3H]TTP in that
same unknown sample can be determined from the tritium content.
FCM. Tumor cells were recovered from Renografin gradients,
washed with balanced salt solution, and analyzed by 2-parameter FCM
using an Ortho ICP 22 (Westwood, Mass.) interfaced with a ModComp
II/25 minicomputer. The same machine settings were used for each
group of samples. Two FCM measurements were made on the cellular
populations of each gradient layer, and a minimum of 10" cells was
analyzed per histogram. One method uses AO to detect differential
sensitivity of quiescent and cycling cells to acid denaturation of the
DNA (3). Aliquots of 106 cells fixed in 70% ethanol were treated with
2000 units RNase for 1 hr at 37°and then placed on ice. The DNA was
partially denatured with HCI, pH 1.5, for 30 sec. Cells were then
stained with AO (S^g/ml), which fluoresces green (530-nm maximum)
when bound to dsDNA and red (640-nm maximum) when bound to
ssDNA. Quiescent cells have been shown to have a greater fraction of
their DNA susceptible to acid denaturation, and therefore, they have a
relatively increased red fluorescence with a concomitant loss of green.
The second method measures RNA:DNA ratios (12). Unfixed cells are
stained with AO (10 fig/ml); green fluorescence is proportional to DNA
content, and the red fluorescence is indicative to RNA content. The
number of cells in any region of the 2-dimension plot is determined by
"box-o-gram"
analysis, which is a computerized method of counting
the number of cells within a given region of the histogram.
Autoradiography.
Cell suspensions were air dried on protein-coated
slides and then fixed with 70% ethanol. The slides were coated with
Kodak NTB2 liquid emulsion at 46°, dried, exposed for 2 weeks, and
then developed
with Kodak D-19 developer.
The background
was 6
grain counts/cell.
The percentage of labeled nuclei was obtained by
counting at least 500 cells/slide.
DNA Content and Specific Activity Determinations.
Total DNA was
measured by the diphenylamine reaction according to the method of
Burton (I). After being labeled with [3H]thymidine, the radioactivity of
the DNA was determined by scintillation counting of an aliquot of the
acid-hydrolyzed sample and a diphenylamine determination of another
aliquot. Under these conditions, only DNA and RNA are hydrolyzed,
thus reducing contamination caused by redistribution of tritium label
into other macromolecules.
RESULTS
Tumor DNA Synthesis. The DNA replication kinetics of FSA
tumor cells was studied by measuring [3H]thymidine incorpo
ration into DNA, TTP pool sizes, and TTP specific activities.
Tumor cells were labeled by i.p. injections of [3H]thymidine,
and after 1 hr, the tumors were removed and subjected to
density gradient centrifugation. The results from each density
band are presented in Table 1. The acid-soluble or unincor
porated label was measured as an estimate of [3H]thymidine
transport or diffusion into each population. The denser cells
contained less label, suggesting that they were less accessible
to the functional vasculature. This same conclusion is reached
if total label (unincorporated and incorporated) is measured.
The acid-insoluble tritium represents incorporation of label
into DNA, and here again, the denser cells showed the lowest
specific activity. To determine whether the reduced rate of
incorporation in the denser cells was due to the inability of
label to reach these cells or to a lower rate of DNA synthesis,
the specific activity of the TTP pools was measured for each
density population using the DNA polymerase assay. As shown
in Table 1, the absolute size of the TTP pool was lowest in the
denser populations, resulting in a higher tritium specific activity.
Therefore, the reduced rate of incorporation in these cell types
was not caused by insufficient label. Because of the high TTP
specific activity, the rate of incorporation in Bands 3, 4, and 5
is an overestimate of the actual rate of DNA synthesis. After
correcting for specific activities of TTP, we determined the
rates of DNA synthesis in Bands 3, 4, and 5 relative to Band 2
to be 23, 22, and 8%, respectively. From the data in Table 1,
it can be concluded that the denser populations, Bands 3, 4,
and 5, are partially isolated from circulating [3H]thymidine and
that these cells, on the average, synthesize DNA at reduced
rates when compared with Band 2.
In Vitro Labeling. FSA tumors were removed and labeled
with [3H]thymidine in vitro before separation on density gra
dients. The incorporation of [3H]thymidine into acid-insoluble
Table 1
In vivo labeling of FSA tumors with [*H}thymidine and TTPpool sizes
la
bel (dpm/jig
(dpm/*ig
Cell
DNA)1
(g/cu
cm)1.061.081.111.141.17Acid-soluble
DNA)313
band12345S-phase
cells
(%)1820182325Density
8.5a150
70 ±
6.6331±
7.5100
±
±10138
6.094
±
7.0150±
.967±1
7.892±
±3.2Acid-insolublelabel
± 3.5TTP
pool (ng
TTP/jig
DNA)50
8.842 ±
4.030 ±
5.117.6±
0.616.8
±
± 2.2TTP
specific
ac
tivity (dpm
[3HJTTP/ng
TTP)12
6.063.9
±
0.617.2
±
1.68.1±
1.213 ±
± 1.6
a Mean ±S.E.
'' TTP pool sizes and specific activity measurements on Band 1 were performed on an insufficient
quantity of material. Therefore, because of the large S.E.. Band 1 has been omitted from the discussion.
5000
CANCER
RESEARCH
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VOL. 42
Kinetics of Density-separated
material was measured, and in addition, samples were taken
for autoradiography. Since high levels of [3H]thymidine incor
poration take place under these conditions, we assumed that
endogenous TIP pools were altered by the added label; there
fore, pool size measurements were not made. Table 2 shows
the results from these experiments. When FSA tumor cells
were labeled in vitro, no significant difference in incorporation
was observed for any of the density populations. A slight
increase in Bands 3, 4, and 5 may have been due to their lower
TTP pools before labeling began. The LI of each band is also
presented in Table 2, and here again, no significant differences
were observed. These results suggest that the same popula
tions that were synthesizing very little or no DNA in vivo are
able to synthesize much greater amounts of DNA under in vitro
conditions. The LI results, also shown in Table 2, suggest that
all density populations incorporate [3H]thymidine in vitro con
sistent with the fraction of those cells that are in S phase as
determined by FCM analysis.
Since the different density populations contain normal cells,
it is important to consider their possible contribution to the
results. Since the FSA tumor cells contain 1.8 times the DNA
of normal cells, the amount of DNA in each fraction contributed
by normal cells can be determined by FCM. The normal cells,
however, do not cycle appreciably (see below), but they do
contribute a small amount of DNA to each fraction. Table 3
shows the percentage of total DNA contributed by normal cells
in each fraction and the results of [3H]thymidine incorporation
after adjusting the results to eliminate the influence of normal
cell DNA. These results when compared with those of Table 1
demonstrate that the differences were small. Since we do not
know the TTP pools of normal cells within tumors, it would be
meaningless to make this adjustment for all the data. Table 3
is presented to illustrate that normal cells probably do not
influence the validity of these observations.
FCM. Chart 1 shows the results of 2-parameter FCM deter
minations of each density population after RNase treatment
and selective acid hydrolysis. After AO staining, ssDNA fluoresces red, while dsDNA fluoresces green. DNA in noncycling,
quiescent cultured cells is more sensitive to acid denaturation
than that in cycling cells and has increased red fluorescence
with a concomitant loss of green fluorescence (3). Chart 1
shows that the fluorescent patterns of all 5 populations were
quite similar. The exception was Band 5, which had a greater
Table 2
In vitro labeling of FSA tumor cells with [3H¡thymidine
label
Cell cm)345.06
band Density (g/cu
(dpm/ng
DNA)4.7
(% of labeled
nuclei)25
±0.4a
.08
.11
.14
.17Acid-insoluble
5.7
7.0
6.9
8.4
±0.4
±0.4
±2.0
±0.3LI
27
24
27
26
Mean ±S.E.
Table 3
Results adjusted for DNA contributed by (he normal cells in FSA tumors
Cell band1
in normal
cells(%)3
2
34
11
9
2
1In
5DNA
DECEMBER 1982
vivo incorporation
(dpm/ng
DNA)323
tumor
372
152
153
93In
vitro incorpo
ration (dpm/ng
tumor
DNA)4.8
6.4
7.7
7.0
8.5
ruse
Tumor Cells
B1
B2
B3
B4
B5
i
88 DNA
-~
Chart 1. Two-parameter FCM pattern of unseparated (USO and separated
(81 to B5) FSA tumor cells, showing the relative sensitivity of chromatin to acid
denaturation. Abscissa, relative amount of ssDNA; ordinate, relative amount of
dsDNA after the cells were treated with RNase and limited acid denaturation as
described in "Materials and Methods." Dots, measurement on a single cell.
Arrow points to the region in which quiescent cells are usually found using this
procedure.
population of G, tumor cells with increased ssDNA. However,
the fraction of noncycling cells in the denser populations can
not be accounted for by this small "quiescent" population; we
determined from cell counts taken before and after fixation that
preferential loss of quiescent cells during preparation did not
occur. Chart 2 shows another set of 2-parameter FCM deter
minations that quantitate the DNA and RNA content of each
cell. Here again, the noncycling population of the denser bands
cannot be differentiated from the cycling cells using this tech
nique. In conclusion, the noncycling cells of FSA tumors gen
erally appear similar to the cycling population and thus far
cannot be selectively identified by FCM. The results shown in
Chart 2 also show that the normal cells were not cycling
appreciably. S-phase normal cells accounted for 2% of the
tumor population at most, as determined by "box-o-gram"
analysis of Chart 2. It can be presumed, therefore, that DNA
synthesis measurements reflect primarily the tumor cell popu
lations.
DISCUSSION
Separation of FSA tumor cells into subpopulations by density
centrifugation has proved to be an excellent system for study5001
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W. A. Brock et al.
the possibility that normal cells contributed significantly to the
DNA synthetic fraction.
It is interesting that all density subpopulations synthesize
DNA at equivalent rates and have the same LI in vitro. Under
these conditions, cells are well nourished and have an equal
availability of labeled precursor. Recovery of the denser (i.e.,
radiation-resistant, hypoxic) fractions apparently is rapid when
the cells are placed under good growth conditions.
Two-parameter
FCM analysis of cells according to the
method of Darzynkiewicz ef al. (3) differentiates between quies
cent and cycling cultured cells, because the chromatin from
quiescent cells is more sensitive to selective acid denaturation
than that from cycling cells. With a possible exception of the
most dense population, the results presented here did not
demonstrate the presence of a "typical" G0 quiescent popu
B1
B3
»B5
Relative DNA Content
—¿
Chart 2. Two-parameter FCM patterns of unseparated (USO and separated
(81 to 85) FSA cells stained with AO. Abscissa, relative DNA contents; ordinate,
relative RNA content of each cell.
ing tumor heterogeneity. Tumors can be treated in vivo or in
vitro before density separation, and then each subpopulation
can be analyzed for clonogenicity, synthesis of macromolecules, cell cycle distribution, radiation and drug sensitivity, or
any number of other parameters.
After an i.p. injection of [3H]thymidine, the cells of heavier
density accumulated label into the soluble fraction at a reduced
rate compared to the lighter cells. This is consistent with the
notion that these cells are in the hypoxic regions of the tumor,
relatively isolated from the circulation. Band 4 cells respond to
y and neutron irradiation in a manner similar to hypoxic cells
(6). These same cells showed a decreased rate of incorporation
of [3H]thymidine into DNA, suggesting that a large fraction of
the cells was noncycling or slowly cycling compared to the
lighter density cells in Band 2. Because the specific activities
of the TTP pools were measured, it is correct to state that the
denser populations are synthesizing less DNA. These conclu
sions are consistent with the LI measurements of Sigdestad
and Ordina (9) that showed a relatively low LI of dense cells 1
hr after [3H]thymidine injection. In addition, FCM measurements
showed that tumor cells in all fractions contained the same
percentage of S-phase cells, thereby ruling out the possibility
that DNA synthesis differences were due to different cell cycle
distributions. Finally, the 2-parameter FCM measurements de
termined that the normal cell population in each density band
accounted for 2% or less of the S-phase cells. This rules out
5002
lation. In fact, it has been estimated that the hypoxic cell
fraction in this tumor is 50% (8), and the appearance of these
cells has not been differentiated from the well-oxygenated,
growing cells by FCM. Another 2-parameter method that mea
sures DNA:RNA ratios also failed to identify the radiation-re
sistant, hypoxic Band 4 fraction of FSA tumor cells. It is
possible that poor growth conditions in the hypoxic regions
simply slowed or stopped the tumor cells in their cycle and did
not provide conditions that would allow them to be identified as
quiescent by FCM analysis. It is important to note, in addition,
that these data show that the hypoxic, noncycling cells in this
tumor can stop progression in all phases of the cell cycle, not
just GL
The technique of acid denaturation followed by AO staining
is very sensitive to cell concentration, quality of fixation, and
rehydration. Small shifts in fluorescence may be artifactual, not
representing a real biological difference between 2 subpopu
lations. We cannot therefore be sure that the higher density
cells do not show a generalized shift in fluorescence that would
indicate a noncycling population.
In conclusion, the FSA tumor system described here contains
a significant hypoxic cell fraction of noncycling or slowly cy
cling cells as determined by studies of DNA metabolism. These
cells can be isolated as a dense cell fraction on density gra
dients of Renografin. FCM measurements do not demonstrate
the significant G0 or quiescent cell fraction that has been
described in cultured cell systems (3). When placed in culture,
the hypoxic cells immediately begin to synthesize DNA at rates
similar to the well-oxygenated cells, presumably because of
improved nutritional conditions. This method of in vivo versus
in vitro labeling may prove useful for identifying the hypoxic
cell fraction of other tumors.
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5003
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Kinetic Heterogeneity in Density-separated Murine
Fibrosarcoma Subpopulations
William A. Brock, Douglas E. Swartzendruber and David J. Grdina
Cancer Res 1982;42:4999-5003.
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