[CANCER RESEARCH 31, 789—797, June 19711
Nucleolar Morphology, Nucleic Acid Syntheses, and Growth Rates
of Experimental Tumors'
Milan PotmesilandAnna Goldfeder
Cancer and Radiobiological ResearchLaboratory, Department of Biology, New York University, and Departments ofHealth and Hospitals, New
York, New York 10032
(densely
stained)
nucleoli
areformed
inproliferative
cellsasin
SUMMARY
Quantitative data are presented showing a relationship
between nucleolar morphology and cell proliferation kinetics
in tumors. Three transplantable mouse mammary carcinomas,
dbrB, IBAH, and IBAH3 , used in this study ranged from
anaplastic to well differentiated. The ratio of their average
growth rate, as measured by the volume and cell-doubling
time, was approximately
1:3:5. Cells with dense nucleoli
predominated in the fast-growing anaplastic dbrB tumor and in
the less-differentiated
IBAH tumor (87.0 and 71.8%,
respectively), as was determined in smear and contact
preparations stained with toluidine blue. Cells with dense
nucleoli, rich in nucleoproteins, exhibited autoradiographically
an extensive incorporation
of uridine-5-3 H. Cells with
ring-shaped nucleoli and cells possessing nucleoli with
well-defined trabeculae predominated in the slow-growing and
well-differentiated
IBAH3 tumor, constituting 50.5% of all
tumor cells. These cells showed autoradiographically a very
low incorporation of uridine-5-3 H.
Kinetic studies were performed with thymidine-methyl-3 H
as a tracer of DNA systhesis. In pulse label experiments, only
31
to
46%
of
cells with
dense
nucleoli
became
labeled.
Repeated injections of the tracer at 4-hr intervals resulted in
labeling of almost all cells with dense nucleoli at 36 hr. At the
same time, cells with ring-shaped and trabeculate nucleoli
constituted the unlabeled cell population. Differences between
kinetic parameters calculated for cells with these types of
nucleoli and cells with dense nucleoli are highly significant.
Results obtained from studies on cell proliferation kinetics
indicate that tumor cells with dense nucleoli represent the
proliferating cell population, whereas cells with ring-shaped
and trabeculate nucleoli constitute the nonproliferating frac
tion in tumors.
nucleoli,
depending
on
the
distribution
of
ribonucleoprotein
structures,
are compact
or contain
well-defined trabeculae (28) or have a ring-like appearance
of cells in tumors.
in
part
(11, 17, 31) and apparently
An extensive
study
was undertaken
to
correlate the frequency of different types of nucleoli with the
growth rate and degree of differentiation in 3 transplantable
mouse adenocarcinomas
propagated
in isologous hosts.
Further, this study is concerned with finding a relationship of
nucleolar morphology to proliferating and nonproliferating
cell populations. This study also constitutes an extension of
previous investigations on structural and biological properties
of tumors of different growth rates (8—10).
MATERIALS
Tumors.
AND
Three
METHODS
types of mouse mammary
carcinomas
were
used. The tumors were carried by serial transplants in
isologous hosts. Specifically, the 200th to 218th passages of
the tumor designated dbrB, the 176th to 187th passages of the
IBAH tumor,
DbrB tumor
Laboratory,
continuously
tumors arose
strain. These
(24).Nucleoli
containing
numerous
trabeculae
orcompactand
1 Supported
A3 treatment
and the 10th to 16th passages of the IBAH3
IBA/Gf strain were recipients of the 2 other types of tumors.
Studies on nucleolar morphology in different cell types have
that
D or chromomycin
reflect a limited rate of RNA synthesis in nucleoli (20, 21).
Thus, it follows that the morphology of nucleoli is related to
the biosynthesis of RNA in the nucleolus.
Several studies have been performed on the morphology of
nucleoli in neoplastic cells, and a comprehensive review on this
subject is available (5). However, information is lacking on the
relation of nucleolar morphology to the proliferative capacity
tumor were used. Young females of the DBA/Gf strain were
recipients of the dbrB tumor implants, and females of the
INTRODUCTION
shown
immature hemopoietic cells (25, 28), in less-differentiated or
undifferentiated
lymphosarcoma
cells (26),
and
in
less-differentiated smooth muscle cells (27). Densely stained
nucleoli are also present in cells with accelerated RNA
synthesis after stimulation with phytohemagglutinin
(21).
Conversely, ring-shaped nucleoli are present in mature and
differentiated cell forms (24, 28, 30) in cells after actinomycin
by
Grant
Ca
0-2565-1
2
from
the
National
Cancer
Institute, NIH, and by grants from the Mildred Werner League for
Cancer Research and from the Anna Fuller Fund.
ReceivedNovember9, 1970; accepted February 5, 1971.
was originally obtained from the Jackson
Bar Harbor, Maine, and since 1964 has been
propagated in this laboratory. IBAH and IBAH3
in this laboratory in female mice of the IBA/Gf
tumors were used for extensive studies (8—10),
their properties
are briefly described
in “Results.―
Measurements of Tumor Growth. The
were grown s.c. When a palpable nodule
of the implant, the size of the tumor
dimensions with calipers. The latent
transplanted tumors
appeared on the site
was measured in 3
period was thereby
JUNE 1971
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789
Milan Potmesil and Anna Goldfeder
Table I
Growth rate of tumors
The differences between the growth rate of all 3 tumor types resulted in a ratio of I :2.9:4.6 (volume-doubling time by
plotting method), 1:3.0:5.1 (volume-doubling time by regression analysis), and 1:2.8:4.6 (cell-doubling time). All values are
mean
S.D.TumorPassagesLatent
±
timeCell-doubling
(days)ing
period
methodRegression
(days)°Volume-doubl
(%)ePlottin@,
analysis―dbrB
a Mean
values
of the
latent
±0.36
3.01 ±0.62
4.84 ±1.040.97
±0.6
8.6 ±0.8
13.9 ±5.61.05
176—185
IBAH
IBAH3200—21810—163.9
period
(time
elapsing
between
the
index
time (days)'@Mitotic
±0.31
2.92 ±0.93
4.96 ±0.840.98
implantation
and
±0.096
2.77 ±0.40
4.47 ±0.151.75
appearance
of
the
the volume
of 0.5
±0.19
0.60 ±0.075
0.37 ±
0.011
first
detectable
to 0.6
cu cm;
tumor
nodule) were estimated for 40 dbrB, 40 IBAH, and 30 IBAHI tumors.
b Plotting
method
was used in 50 dbrB,
40 IBAH,
and
12 IBAH3
tumors
reaching
the slope
of the growth line was constructed with the use of the logarithms of tumor volumes at different days after implantation.
CRegression
analysis
was
used
in40dbrB,
30IBAH,
and
12IBAH3
tumors
reaching
the
volume.of0.5
to0.6
cucm;
the
equation for the regression lines
log (10 X volume in cu cm) a + b@0iX days
was used in calculation of the rate constants for the growth in volume (b@01);volume-doubling time (in days) = 0.30l/b@01
(16). The coefficient of correlation between the volume-doubling time estimated by the plotting method and by the regression
analysis is significant (dbrB andlBAH,p < 0.05;IBAH3, p < 0.01).
d Mean
values
of the
cell-doubling
time
were
determined
with
the
use of the
rates
of accumulation
of mitoses
in 10 dbrB,
10 IBAH, and 6 IBAH3 tumors reaching the volume 0.5 to 0.6 cu cm 6 hr after colchicine treatment; cell-doubling time (in
days) = 100/% mitoses/day (16). The values of cell-doubling time and volume-doubling time obtained for individual tumors
were compared with the use of the standard deviation of the measurements in pairs (dbrB tumors, a ±0. 11; IBAH tumors, a ±
0.38; IBAH3 tumors, a ±0.61).
e Cells
in mitoses
were
determined
viewing
1000
cells in each sample.
For
the estimation
of mitotic
index,
smear
preparations of 10 dbrB, 10 IBAH, and 6 IBAH3 tumors were used.
ascertained. The volume of tumors was calculated in cu cm by
the formula
v=
(ir/6)
X D1 X D2 X D3
in which D is 3 different diameters in mm. These
measurements
were used for the evaluation
of the
volume-doubling time. The average growth in volume was
expressed by the slope of the line obtained from the
logarithms of tumor volumes, calculated at different days after
implantation. The slope was used for the estimation of the
volume-doubling time for individual tumors of 0.5 to 0.6 cu
cm volume by the plotting method. The volume-doubling time
was also calculated for tumors of corresponding sizes by
regression analysis with the equation for regression lines (14):
log(lO X volume)
a + b@01X days
The cell-doubling time was determined in all 3 types of
tumors by the colchicine method (3, 4, 14). Volume
measurements of these tumors were made, and the volume and
cell-doubling times were compared. Mice bearing tumors
approximately 0.5 to 0.6 cu cm in volume were given i. p.
injections at 10:00 a.m. of 0.02 mg colchicine/20 g body
weight and killed 6 hr later. Peripheral translucent portions of
tested tumors were fixed in Bouin's solution and embedded in
paraffin. Histological sections were cut at 3 p and stained with
hematoxylin and eosin. The proportions between dividing and
nondividing cells were estimated on the basis of viewing 1000
mitoses in histological sections of each tumor. Results were
expressed as the percentage of mitoses/day. The cell-doubling
time was determined in days, by the formula lOO/(%
790
mitoses/day) (14). Preliminary tests were made to estimate the
optimal dose of colchicine and the optimal time intervals of its
action. The results revealed no diurnal fluctuation of mitotic
activity within the 24-hr period.
Techniques Used For Light Microscopy. When the volume
of the tumor implants reached 0.5 to 0.6 cu cm, mice were
sacrificed by disconnection of the spinal cord. The tumor
tissue was immediately removed, and peripheral portions of
the tumor were used for smear and contact preparations. The
preparations were dried in air and stained with a 0.05%
solution of buffered toluidine blue at pH 5.0 without previous
fixation (24, 30). This standardized procedure demonstrates
structures containing RNA to a high degree of specificity (29).
In addition, small pieces of tumor tissue were fixed for 24 hr
in 4% neutral formaldehyde solution and routinely processed.
Tissue sections cut 5 p thick were stained in 0.1% solution of
toluidine blue (6). Small pieces of several tumors were fixed in
Bouin's fixative, routinely processed, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin for the
study of the overall morphology.
For autoradiographic
studies, UrR-3 H2 and TdR-3 H
( Schwarz
BioResearch,
Inc.,
Orangeburg,
N.Y.)
were
used
as
tracers. Injections of a single dose of UrR-3 H (2 pCi/g body
weight; specific activity, 28.0 Ci/mM) were given to a
representative number of mice bearing a specific tumor type.
These mice were sacrificed 30 mm later. This time interval was
found to be optimal for an efficient labeling of nucleoli in all 3
types of tumors. In another experiment, tumor-bearing mice
2The abbreviationsused are: UrR-3H, uridine-5-3
H; TdR-3H,
thymidine- methyl-3 H.
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@
!@
@
2@
I
3@
Nucleolar Morphology and Growth Rates of Tumors
@
@
were given injections of TdR.3 H (1 uCi/g body weight: soecific
activity, 1.9 Ci/mM) and sacrificed 30 mm later. For con
tinuous labeling, mice bearing dbrB and IBAH tumors were
given injections of TdR-3 H (0.5 pCi/g body weight) at 4-hr
intervals for periods of 12, 24, and 36 hr (2). Smear
preparations of labeled tumors were covered with Kodak
liquid NTB2 photographic emulsion and processed by the
established method. The necessary exposure time of prepara
tions with UrR-3 H label was 120 days; with TdR-3 H label it
was 10 days. In addition, several preparations with TdR-3H
label were exposed for 175 days. Smear preparations were
stained with toluidine blue after developing (20).
Criteria and Statistical Evaluation. The different types of
nucleoli were determined in cells of the 3 types of tumors with
smear and contact preparations stained with toluidine blue.
Nucleoli were classified with respect to their morphology as
ring-shaped nucleoli, nucleoli with well-defined trabecular
structures, and densely stained (compact) nucleoli (24, 28).
The 2 latter types of nucleoli are referred to, in brief, as
“trabeculatenucleoli―and “densenucleoli,―respectively. The
results were expressed as the percentage of cells possessing
different types of nucleoli. Cumulative curves of percentages
were constructed for samples of each tumor. These curves
served to deduce the statistically significant number of cell
counts (22). The range of variation in samples of each tumor
was on the order of 10% and decreased to less than 5% before
reaching the count of 1000 cells. This number of cells was
considered statistically significant for the evaluation of
samples.
In quantitative autoradiographic studies, silver grains were
counted above 500 cells of each sample labeled with UrR-3 H
and above 600 or 1000 cells of each sample labeled with
TdR-3 H in smear and contact
statistical
purposes,
monolayer
preparations.
the number of grains counted
IBAH
@Ioo 10x103dbrB
IBAH3
For
in each
-6xl03
z
GRAIN COUNT OVER NUCLEOLI PER CELL
Ioo@
5O@
dbrB
RSN 0
TN
9
30@
go-I 6
IJ
I
dbrB
50
30
‘?
I
IBAH
I00
CELLS
00
$00
IBAH
50
30
II@
IBAH3
I00
00
,@
:
IBAH3
RSNO7
TN 2
DN 94
50
30
50
30
0
I
0
I
I
I
10
20
30
NUMBEROF GRAINS
1
40
NUMBEROF GRAINS
Chart 2. Incorporation of UrR-3 H in cells of 2 dbrB, 2 IBAH, and 2
IBAH3 tumors (a- - -0, cells with ring-shaped nucleoli; X- - -x , cells with
trabeculae nucleoli; .-.----., cells with dense nucleoli); 500 cells were
evaluated for the grain count and nucleolar morphology in each sample.
The background corresponds to 0.39 grain/100 sq @i,Mean numbers of
grains over cells with ring-shaped nucleoli (RSN), over cells with
trabeculate nucleoli (TN), and over cells with dense nucleoli (DN) are
included in the chart.
sample was higher than 1700 ( 1). The labeling of cells with
different types of nucleoli was evaluated, and a statistically
significant number of counts of cells was deduced with the
previously described method of cumulative curves. The effect
of background was taken into consideration in statistical
evaluation, with the use of grain count distribution curves
(19), Stillstrom's method for calculation of the fraction of
labeled cells (1 ), grain count over “empty―
spaces without cell
structures or debris, and, in preparations pulse labeled with
TdR-3 H, number of grains per mitotic figure (1).
w
I-.
Ui
0
.
C,)
4
:,
z
Ui
I-
@
GRAINCOUNT
PERCELL
CELLS
50
5xI03
-3x10
.j
Results obtained for all 3 types of tumors were evaluated
and compared on the basis of the confidence intervals of the
sigma values
and median
values, the correlation
coefficient,
and Student's t test (7, 18).
0
w
U)
z
U)
ID
4
Ui
0
Ui
a.
.
-I
-I
Ui
0
U)
-J
-J
Ui
C-)
(°)
(b)
(C)
(a) (b) (c)
(a) (b) (C)
-0
Chart 1. The incidence of cells with different morphology of nucleoli
RESULTS
Morphology of Tumors. The morphology of tumors was
evaluated according to their relative degree of differentiation.
Examination of tissue sections stained with hematoxylin and
eosin revealed in dbrB tumors sheets of epithelial cells rich in
cytoplasm (Fig. la), in IBAH tumors nests of cells with
discrete acini forms in some areas (Fig. 1b), and in IBAH3
in dbrB, IBAH,and IBAH3 tumors. a, cellswith ring-shapednucleoli; b,
cells with trabeculate nucleoli; c, cells with dense nucleoli. Columns, tumors epithelial cells arranged in nests and multiple rows
mean values obtained from 10 dbrB, 10 IBAH, and 6 IBAH3 tumors. (Fig. 1c). Thus, these tumors range in morphology from the
anaplastic dbrB tumor to the more-differentiated
IBAH and
Vertical bars, S.D. ; left scale, absolute number of cells evaluated in
IBAH3
tumors.
dbrB and IBAH tumors; right scale; absolute number of cells evaluated
in IBAH3 tumors. Differences in the values of a, b, and c among all
Growth Rate of Tumors. The data obtained from the
types of tumors are highly significant as evaluated with the t test measurements of the latent period, volume-doubling time, and
(P—0.00l).
cell-doubling time of each tumor type are presented in Table
JUNE
1971
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791
Milan Potmesil and Anna Goldfeder
@
@
@
@
IBAH3 tumors, stained with toluidine
@
d br B
1. The correlation
between the volume-doubling
time
estimated by the plotting method and by regression analysis is
significant in all 3 types of tumors (p < 0.05). This correlation
indicates that the growth of tumors was nearly exponential.
The correlation between the values of the cell-doubling time
and volume-doubling time of all 3 tumor types is significant as
indicated by the standard deviation of the measurements in
pairs. The differences between the volume-doubling and
cell-doubling times of dbrB, IBAH, and IBAH3 tumors are
approximately in a ratio of 1:3:5.
The latent period and the volume-doubling time of 10 dbrB,
10 IBAH, and 6 IBAH3 tumors, which were used for studies of
nucleolar morphology, correspond to those presented in Table
1, and the difference between the volume-doubling time
resulted also in a ratio of 1:3:5.
Morphology of Nucleoli. Ring-shaped nucleoli with an
intensely stained peripheral area (Fig. ld), trabeculate nucleoli
with trabecular basophilic structures distinctly separated by
light areas (Fig. le), and dense nucleoli nearly uniformly
stained (Fig. 1/) were present in histological sections of dbrB,
IBAH, and IBAH3 tumors stained with toluidine blue. The
distribution of ribonucleoproteins of nucleoli in histological
sections corresponded to the observations made in smear and
contact preparations.
In smear and contact preparations, cells of dbrB, IBAH, and
400
z
Ui
U)
4
-J
-I
Ui
I00-@
-
RSN, TN
..o--
0
cy'-@ Lcb.I.d
0
30mm
I2hr
ca//s
24hr
36hr
TIME
IBAH
800
Ui
ID
400
Ui
3
200
U) 100
nucleoli. Among these, nucleoli with a light central area and
basophiic structures in the periphery were observed in cells
with distinct cytoplasmic basophiia (Fig. 2a) and in cells with
a narrow rim of cytoplasm (Fig. 2b). Trabeculate nucleoli
possessed
well-defined
trabecular
basophiic
structures
separated by light areas (Fig. 2c). Nucleoli of this type ranged
from 1 to 3 /2 in diameter. Dense nucleoli ranged from those
almost uniformly stained to those with more or less distinct
small, light areas. The size of dense nucleoli varied from 3 to 7
p in diameter, and their shapes were round, oval, or irregular
(Fig. 2d).
IBAH3
300
ID
z
IBAH
I.
Ui
ID
1 to 6
dbrB
blue, possessed
800
aba/ad ca//s
300
U)
ID
4
U)
-J
200
RSN, TN
00
-j
Ui
0
0'#_
—
Lobe/ad
ca//s
I0
30mm
I2hr
24hr
36hr
TIME
Chart 4. Continuous
labeling of dbrB and IBAH tumors with
TdR-3
H.DN,cells
withdense
nucleoli;
RSN,
cells
withring-shaped
3 xIO
nucleoli;
TN,
cells
with
trabeculate
nucleoli;
1000
cells
were
evaluated
in each tumor.
Ui
-2@I0@
-IxIO
-I
Ui
0
0-
-0
Chart 3. Pulse labeling of dbrB, IBAH, and IBAH3 tumors with
TdR-3H. a, cells with ring-shaped nucleoli; b, cells with trabeculate
nucleoli; c, cells with dense nucleoli; dotted area, pulse-labeled
cells.
Columns, mean value obtained from 5 tumors; 600 cells were evaluated
in each tumor. Vertical bars, S.D. TdR-3 H was injected 30 mm before
sacrifice.
792
For quantitative evaluation of nucleoli, smear and contact
preparations stained with toluidine blue were used (3 1). Cells
of all tumor types were divided into 3 groups according to
their nucleolar morphology: (a) cells with ring-shaped nucleoli;
(b) cells with trabeculate nucleoli (more than two-thirds of
these cells also possessed ring-shaped nucleoli); and (c) cells
with dense nucleoli (one-third of these cells also possessed
ring-shaped nucleoli, trabeculate nucleoli, or nucleolar frag
ments). Only intact tumor cells were used for this study;
necrobiotic tumor cells, cells of the tumor stroma, and
infiltrating cells were excluded.
In dbrB tumors, cells with ring-shaped nucleoli constituted
1.67%, and cells with trabeculate
nucleoli
constituted
1 1.3 1%
in IBAH tumors values were 7.03 and 21 .12%, and in IBAH3
tumors they were 15 .42 and 35 .08%, respectively. Cells with
dense nucleoli were present in 87.02% of the dbrB, 7 1.85% of
the IBAH, and 49.50% of the IBAH3 tumors (Chart 1).
Nucleolar Morphology and RNA Synthesis. The UrR-3H
CANCER
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Nucleolar Morphology and Growth Rates of Tumors
labeling was studied in cells of 5 dbrB, 5 IBAH, and 3 IBAH3 nucleoli were observed among different types of tumors. The
tumors. The distribution
of silver grains was compared highest number of heavily labeled cells or of heavily labeled
between cells with different types of nucleoli. After a 30-mm nucleoli was observed in dbrB tumors; the lowest was observed
pulse, most of the grains were located over dense nucleoli and in IBAH3 tumors.
Nucleolar Morphology and DNA Synthesis. The initial
over nucleoplasm of cells with dense nucleoli (Fig. 2e).
Ring-shaped and trabeculate nucleoli showed very low or no labeling index was estimated in all 3 types of tumors after a
labeling, and the incorporation of the label in nucleoplasm of single injection of TdR-3 H. Pulse labeling for 30 mm resulted
cells possessing these types of nucleoli was very low. The in 39.4% of labeled cells in dbrB tumors, 22.1% of labeled cells
quantitative evaluation of the label was done in 2 tumors of in IBAH tumors, and 13.2% of labeled cells in IBAH3 tumors
each tumor type. The grain counts over cells and over nucleoli (Chart 3). A portion of cells with dense nucleoli was labeled
were recorded as grain count distribution curves (Chart 2). The (Fig. 2/), representing less than one-half of all cells with dense
mean number of grains over cells with ring-shaped nucleoli is nucleoli in dbrB tumors and less than one-third in IBAH and
approximately
13 to 18 times lower, and over cells with IBAH3 tumors. Cells with ring-shaped nucleoli and cells with
trabeculate nucleoli it is 7 to 9 times lower than the mean trabeculate nucleoli remained unlabeled. These cells remained
number of grains over cells with dense nucleoli. Differences in unlabeled even in preparations exposed for 175 days.
the extent of UrR-3 H incorporation in cells with dense
Repeated injections of TdR-3 H at 4-hr intervals increased
the number of labeled cells at 36 hr to 94.4% in dbrB tumors
I000j
and to 84.5% in IBAH tumors. The percentage oflabeled cells
/84/1 - DN
with dense nucleoli gradually increased ; at 36 hr, nearly all
cells with this type of nucleoli were labeled (Chart 4). Labeled
cells with ring-shaped or trabeculate nucleoli appeared in 1.8%
of the whole cell population in dbrB tumors and in 2.2% in
Ui
ID
IBAH tumors at 12 hr after continuous labeling. An increase
to 5.6 or 8.6% was observed at 36 hr. At this time interval,
D
z
I00
Ui
I-
7.6% of the entire cell population
of
@1
0U)
TN
whole
cell
population
in dbrB tumors and 15.5%
in
IBAH
tumors
remained
unlabeled. All unlabeled cells in dbrB tumors were either cells
with ring-shaped nucleoli or cells with trabeculate nucleoli. In
ID
4
Cl)
-i
IBAH tumors, in addition to these cell types, a small portion
-J
Ui
C-)
the
I0
30mm
24 hr
I2hr
36hr
TIME
Chart 5. Decrease of unlabeled cells in continuously labeled dbrB
and IBAH tumors. DN, cells with dense nucleoli; RSN, cells with
ring-shaped nucleoli; TN, cells with trabeculate nucleoli.
of cells with dense nucleoli (approximately 6%) remained
unlabeled.
For establishment of proliferation kinetic parameters of
cells with dense nucleoli and cells with ring-shaped and
trabeculate nucleoli, the kinetics of TdR-3 H incorporation was
evaluated as 50% decrease of unlabeled cells (Chart 5), labeling
increment, efflux from the unlabeled pool of cells, and average
transit time (Table 2). The labeling increment ( 13) of cells
with dense nucleoli indicates the fraction of proliferating cells
which has entered DNA synthesis during a certain time
interval. The labeling increment of cells with ring-shaped and
Table2
Kinetic parameters of cells with different
types of nucleoli
All parameters were calculated with data presented in Charts 4 and 5.
from the
increment
2dbrBCells
(cells/hr/
TumorLabeling
(hr)Method
transit timea
unlabeled pool
30 min—36hr
100 cells)Efflux (cells/hr/
100 cells)Average
1Method
nucleoli1.371.49Cells
with dense
ring-shaped0.160.1865.571.2nucleoli
with
trabeculatenucleoliIBAHCells
and
nucleoli1.431.66Cellswithring-shaped0.240.2473.273.7nucleoli
with dense
trabeculatenucleoli
and
a Calculated
with
the
plotting
method
(Method
1) and
the
formula
% nonproliferating
cells/efflux
of
unlabeled cells (Method 2) ( I 3).
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793
Milan Potmesil and Anna Goldfeder
trabeculate
nucleoli is significantly
with dense nucleoli.
lower compared to cells
It seems reasonable
to assume that their
labeling increment may be interpreted rather as an efflux of
labeled cells from the proliferating compartment. Similarly,
the efflux from the unlabeled pool (1 3) is lower in the
compartment of cells with ring-shaped and trabeculate nucleoli
and may indicate a replacement of unlabeled cells in this
compartment. Differences between 2 compartments of cells
are further indicated by the 50% decrease of unlabeled cells.
This decrease proceeds more rapidly in the compartment of
cells with dense nucleoli as was noted in dbrB and IBAH
tumors.
DISCUSSION
This study demonstrates that cells of mouse mammary
adenocarcinoma contain different types of nucleoli such as
dense nucleoli, nucleoli with well-defined trabeculae, and
ring-shaped nucleoli, as were observed in smear and contact
preparations of the tumor tissue. Tumor cells with ring-shaped
nucleoli and cells possesssing nucleoli with well-defined
trabeculae showed a very low incorporation of UrR-3 H as
noted autoradiographically;
this correlated with previous
observations on other cell types (20, 2 1). Conversely, an
extensive
incorporation
of
UrR-3H
was
noted
autoradiographically
in the tumor cells with dense nucleoli.
This is in accord
with
an observation
that the presence
ACKNOWLEDGMENTS
of
large, dense nucleoli, rich in ribonucleoprotemns, indicates
rapid synthesis of ribosomal precursors (20, 21). A portion of
tumor cells possessed ring-shaped nucleoli in addition to dense
nucleoli. This indicates that “resting―
and “active―
nucleoli
can be present in the same nucleus of a tumor cell. Such an
observation was also made in immature leukemic cells (25).
The above results are consistent with previous findings that
the distribution of ribonucleoprotein structures in nucleoli
indicates metabolic activity leading to cell proliferation, as was
observed in vivo (24, 25, 28) and under experimental
conditions in vitro (15, 20, 21). For more conclusive
information on this aspect, experiments with thymidine-3 H
label were performed. As shown in Charts 3 and 4, pulse
labeling of tumors with TdR-3 H resulted in the labeling of a
certain portion of cells with dense nucleoli, and continuous
labeling for 36 hr resulted in the labeling of nearly all tumor
cells with dense nucleoli. This indicates that cells of this type
were synthesizing DNA for further cell division. Conversely,
cells with ring-shaped or trabeculate nucleoli were not pulse
labeled, and a considerably high portion of these cells had not
entered into DNA synthesis within 36 hr of continuous
labeling and remained unlabeled at this time. The low
percentage of labeled cells with ring-shaped or trabeculate
nucleoli within 12 and 36 hr may be interpreted as an efflux
from the proliferating compartment into the nonproliferating
one. As indicated in Table 2, estimates from the increments in
labeling index due to serial TdR-3 H injections were calculated
per time unit for cells with different types of nucleoli.
Differences between kinetic parameters established for cells
with dense nucleoli and for cells with ring-shaped or trabeculate
nucleoli are highly significant. It follows that cells with dense
794
nucleoli are metabolically active and proliferating and can be
regarded as constituting the “growthfraction― of tumors (16,
23). Cells with dense nucleoli predominated significantly in
the fast-growing anaplastic dbrB tumor and, to a lesser extent,
in IBAH tumor, the latter being differentiated and of a slower
growth rate. Conversely, tumor cells with ring-shaped nucleoli
and cells with trabeculate nucleoli predominated in the slowly
growing and well-differentiated IBAH3 tumor.
The results have shown that the presence of ring-shaped and
trabeculate nucleoli indicates a repressed biosynthesis leading
to the cell growth and the cell division. This is consistent with
the interpretation that tumor cells with these types of nucleoli
represent the nonproliferating
cell population. Moreover,
quantitative data on the incidence of cells with ring-shaped
nucleoli in the well-differentiated IBAH3 tumor suggest that a
major portion of cells in this tumor is “mature―
and
differentiated. Ring-shaped nucleoli are generally present in
mature and differentiated cells of different origins (24—28).
This study established a correlation between nucleolar
morphology, nucleic acid syntheses, and growth rates of 3
types of isologous mammary carcinomas. The methods and
criteria used may prove useful for studies of proliferating and
nonproliferating
tumor cells and for evaluation of the
effectiveness of exogenous agents on tumor growth.
We acknowledge the skillful technical assistance of Miss D. F.
Johnson, Mr. A. K. Ghosh, and Mr. R. L. Timbers.
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1971
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795
Milan Potmesil and Anna Goldfeder
Fig. I . Histological sections: a, dbrB tumor with sheets of epithelial cells rich in cytoplasm; b, IBAH tumor showing nests of cells with discrete
acini forms in some areas; c, IBAH3 tumor with epithelial cells arranged in nests and rows. H & E, x 625. d, cell with ring-shaped nucleolus (arrow)
from an IBAH3 tumor; e, cell with trabeculate nucleolus (arrow) from a dbrB tumor; f, cell with dense nucleolus (arrow) from a dbrB tumor.
Toluidine blue, X 2000.
796
CANCER
RESEARCH
VOL. 31
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1971 American Association for Cancer Research.
Nucleolar
Morphology
and Growth Rates of Tumors
2
7
,
‘7-
@-,
@4
•1 • -
@
•
@.
@
.
@
.
,/,@@
@‘
,
‘@‘l@
Fig. 2. Smear preparations: a, cell with intensely stained cytoplasm possessing ring-shaped nucleolus (arrow) from an IBAH tumor; b, cells with
a narrow rim of cytoplasm possessing ring-shaped nucleoli from dbrB tumor; c, cell with trabeculate nucleoli (arrows) from an IBAH3 tumor; d,
cell with dense nucleolus (arrow) from a dbrB tumor. Autoradiography of smear preparations: e, cell with dense nucleoli (arrows), silver grains
over nucleoli and nucleus (dbrB tumor labeled with UrR-3H); f, cells with dense nucleoli; silver grains over nuclei (dbrB tumor pulse labeled with
TdR-3 H). Toluidine blue, X 2000.
JUNE
1971
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797
Nucleolar Morphology, Nucleic Acid Syntheses, and Growth
Rates of Experimental Tumors
Milan Potmesil and Anna Goldfeder
Cancer Res 1971;31:789-797.
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