[CANCER
RESEARCH
28, 724-725, April 1948]
The Mitotic Cycle of Sarcoma 1801
Linda Simpson-Herren, Jane G. Blow, and Radette H. Brown
Kettering-Meyer
Laboratory,2 Southern Research Institute,
Birmingham,
SUMMARY
The mitotic cycle of proliferating cells in Sarcoma 180 solid
tumors was analyzed by following the incidence of labeled mi
toses with time. The method utilized a dispersed cell prepa
ration treated to demonstrate mitotic figures. The average
lengths of the cycle and its phases in hours were: total cycle,
13.5; S, 7.8; G2, 2.5; GÌ,
2.7; mitosis, approximately 0.5.
INTRODUCTION
In view of the recent interest in the relationship between
sensitivity of cells to various chemotherapeutic agents during
specific phases of the mitotic cycle, it seemed worthwhile to
develop a technic for determination of the cycle in solid tumors
which would yield a representative sample of the cell popula
tion and would be usable for routine analysis. In preliminary
work in our laboratory, we found that mitotic activity and
percent labeled cells following administration of TdR-3H
varied several-fold from one area of a tumor section to another
and also between widely separated sections of the same tumor.
The difficulties of selecting representative areas for scoring
large numbers of interphase and mitotic cells to utilize the
technic of labeled mitotic waves (1) led to the use of a dis
persed cell preparation treated to demonstrate anaphase-metaphase figures. The results of studies of S-180,3 a solid tumor
used extensively for screening of potential anticancer agents,
are reported here.
MATERIALS AND METHODS
In each of the experiments, tumors were used five days after
subcutaneous trocar implantation of a 20- to 30-mg tumor frag
ment into the axillary region of Swiss female mice. At the time
the animals were sacrificed, the tumors ranged in weight from
0.18 to 0.95 gm with the exception of one tumor, which weighed
1.56 gm. TdR-3H (New England Nuclear Corporation, 6.7 c/
1 This investigation was supported in part by Contract PH4366-29 with the Cancer Chemotherapy
National Service Center,
National Cancer Institute, NIH, Bethesda, Maryland, and in part
by funds made available by Southern Research Institute.
2 Affiliated with Sloan-Kettering Institute for Cancer Research,
New York, N. Y.
s The abbreviations used are TdR-3H, thymidine-methyl-3H ;
S-180, Sarcoma 180; Tc, length of cell cycle in hr; Ta, length of
S-phase in hr; TQ , length of Gj in hr; Tg length of G2 in hr;
TM, length of mitotic period in hr.
Received September 25, 1967; accepted December 28, 1967.
724
Alabama 35205
mmole) was administered intraperitoneally at 2 w/gm body
weight, and three mice were killed by asphyxiation with carbon
dioxide at each time period for 21 or 31 hr. The 3 tumors
from each group were pooled, finely minced, and suspended
in 10 ml of 0.25 M sucrose (if total weight of tissue was 1 gm
or less) or in 9 ml per gram of wet tissue (if the weight of
tissue exceeded 1 gm). The cells were dispersed by 3-5 strokes
of a teflon pestle in a glass homogenizing vessel with a clearance
of 0.006-0.009 inches. The suspension was filtered through
four layers of surgical cotton gauze or, preferably, through
a fine wire mesh filter (approximately 0.4 mm openings), to
remove remaining clumps. The filtered cell suspension was
then centrifuged for 5 minutes at 450 X g (at the tip of the
40 ml conical). Two methods were used for processing the
packed cells, but all slides in a given experiment were processed
by the same method. For the first method, which is similar to
that used by Coons et al. (4) for in vitro studies of human
tumor tissue, 5 ml of cold 0.9% sodium citrate was added with
continuous stirring to a 0.2-ml aliquot of the packed cell resi
due and, while mixing, 5 ml of freshly prepared fixative (2:1
ethanol:acetic acid) was added dropwise. This suspension was
centrifuged for 5 minutes at full speed of a clinical centrifuge.
The supernatant was decanted and the cells resuspended in
about 1 ml of fixative. Four to 5 drops of the sample were
placed on a cleaned microscope slide previously moistened with
50% ethanol and the solution was ignited. After thorough dry
ing, the slides were either coated with photosensitive emulsion
or held for later processing. For the alternative method, which
is an adaptation of the procedure used by Puck et al. (8) for
study of cells in culture, 4 ml of cold 0.1 M citric acid was
added to a 0.2-ml aliquot of the packed cell residue and mixed
well. The sample was warmed with continuous agitation in a
37°C water bath for 30 seconds and centrifuged for 5 min at
half-speed in a clinical centrifuge. The supernatant was de
canted and the residue resuspended in about one ml of fixative
(3:1 ethanol:acetic acid). After the sample was chilled in an
ice bath for 4 min, a 0.1-ml aliquot was applied to each slide
(usually from 6 to 12 slides) and allowed to air-dry. Dry slides
were placed in l M HC1 (60°C) for 1 min and again air-dried.
Slides were held for later processing or coated with emulsion
immediately.
Cells processed by the second method gave better radioautographs because the nuclei were flatter and the slides contained
less debris; however, the mitotic figures were not as easily
recognized. In more recent work, saline (0.85%) was used in
stead of sucrose to suspend the minced tissue, and when used
with the second technic for displaying mitotic figures, yielded
satisfactory slides.
CANCER RESEARCH VOL. 28
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The Mitotic Cycle of Sarcoma 180
The calculations are similar to those used by other investi
gators (1) except for the determination of cell cycle time, Tc,
which was calculated as the time between the midpoint of the
ascent of the first peak and the midpoint of the ascent of the
second peak (11). This method avoids the necessity for desig
nating the midpoints of plateaus of irregularly shaped peaks
that are probably due to variations in the time required by
various cells to pass through parts of the cycle. The time of
S-phase (T8) was measured from the midpoint of the first
ascent to the midpoint of the first descent, and the length of"
G2 (TQ2) is the time between injection of the labeled thymidine and the midpoint of the first ascent. The length of mi
tosis (Tu) is calculated from the formula given in Table 1
(8). The formula is based upon the assumption that during
exponential growth the cell distribution through the cycle is
logarithmic rather than linear; that is, at any given moment
there are more cells in the earlier part of the cell cycle than
in the latter part. The mitotic index of these tumors was so
RESULTS
variable that the values given are estimates based on the aver
The results of two experiments utilizing dispersed cell prepa
age mitotic index for each experiment. The length of Gt is
equal to TC-(T8+TO2+
TM).
rations are presented in Chart 1 and Table 1. The agreement
of results would indicate that the length of the cell cycle did
The cell cycle of only the proliferating population of the tu
not change over the 2-month interval (8 serial passages) be
mor is measured by the technic of labeled mitotic waves. Any
tween the experiments and that analysis of the cycle by this nonproliferating (G0) cells would not affect the calculated cycle
time. An average of 34% of the cells in a 5-day-old S-180
method is reproducible.
were labeled by TdR-3H after a one-hr exposure and 86%
were labeled following 8 injections of TdR-3H (total dose 2
Mg/gm body weight) given 3 hr apart over a period of approxi
mately 1.5 average cell cycles. The remaining 14% of the popu
lation may be intact dead cells, nonproliferating (G0) cells, or
cells with cycle times significantly longer than average. An
other possible explanation might be that a proliferating seg
ment of the population lacks thymidine kinase and would not
utilize thymidine for synthesis of DNA. The fact that 14%
of the mitotic figures are unlabeled following multiple injections
of TdR-3H is consistent with the latter possibility but further
evidence will be necessary before a choice can be made between
the possible explanations.
14
18
22
The percentage of cells (34%) labeled by a pulse exposure
Hours after TdR-3H
to thymidine-3H is lower than would be predicted by calcula
Chart 1. Incidence of labeled mitoses in solid Sarcoma 180 tion using the exponential equation given by Cleaver (3) for
determination of the labeled fraction. From the expanded form
versus time in hours after injection of thymidine-methyl-3H
(TdR-:iH). The tumor line was transplanted serially 8 times be
of the equation, the first three terms yield an estimate of 53%
tween Experiment 1 (O
O) and Experiment 2 (X
X).
labeled cells, if all cells were in cycle at the time of the pulse
label. Preliminary results from experiments now in progress
Table 1
indicate that the low experimental value may be attributed to
an even smaller proliferating population than would be indi
(appro*.)M0.50.5054.0\3.02.52.820.0
«22.52.52.518.5T
cated by the thymidine index following multiple injections.
The dose level of TdR-3H used, 2 M/gm, is within the
1Experiment
Experiment
2Average%
range of 1.0 to 10.0 /*c/gm body weight that Lisco et al. (6)
found decreased the number of Ehrlich ascites cells in vivo
of cycleTo141313.5Ts87.57.857.5T
after periods of 5 to 12 days. However, other experiments in
Length of various phases of the mitotic cycle of Sarcoma 180. this laboratory indicate that TdR-3H injected intraperitoneally
Tc, length of mitotic cycle in hr; T8, length of S-phase in hr; TQ ,
into mice is more readily utilized by ascites cells than by solid
length of G2 in hr; To length of Gx in hr; TM, length of mitosis
tumors. Four- to six-week exposures of the emulsion covered
slides were necessary to yield grain counts of 15-40 grains per
Tr log (1 + MI)
in hr =
where MI is the mitotic index.
cell. Most of the calculations are based on results obtained
0.301
Radioautographs were prepared by dipping the dry slides
in a 30% solution of Ilford Nuclear Research Emulsion Type
K-2 (Ilford Limited, Ilford, Essex, England) at approximately
45°Cand air-drying prior to storing at 4°Cfor 4-6 weeks with
one or more standard slides (2) ; the emulsion-covered slides
were then developed in Kodak Developer D-19. The nuclei
were stained through the emulsion with 0.06% Toluidine blue
in 0.2% borax solution (10) and destained by dipping in
ethanol. Coverslips were mounted with Canada balsam and the
slides were viewed under oil immersion. Three thousand cells
were observed on each slide and scored as labeled or unlabeled
interphasc, and labeled or unlabeled mitotic. All clearly recog
nizable anaphase and metaphase figures were classified as mi
totic and, in the event of a very low mitotic index, the slide
was scanned until at least 25 mitotic figures were found and
scored. The mitotic index was usually in the range of 1.5 to 2%.
APRIL 1968
725
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Linda Simpson-H erren, Jane G. Blow, and Paulette H. Brown
during the first 15 hr following exposure to TdR-3H which
allows little time for radiation damage to accumulate. The short
in vivo exposure combined with low incorporation of TdR-3H
makes it likely that radiation damage did not significantly
influence the length of the mitotic cycle.
DISCUSSION
The Tg of 13.5 hr for S-180 is about the same as the 17-hr
generation time of fibrosarcoma (5) and the 15.7-hr generation
time found by Wheeler et al. (11) in L1210 S cells grown
both in the ascites form in vivo and in cell culture, and nearly
one-half of the 34-hr generation time of C3H spontaneous mam
mary tumors studied by Mendelsohn (7). Unpublished results
from our laboratory would tend to support the theory that
the mitotic cycle is characteristic of the tumor line and does
not vary significantly with age and size of tumor or volume
of ascitic fluid.
Knowledge of the length of the cell cycle and the length of
G!, S, G2, and M may be used to advantage in planning
schedules for treatment of S-180 with agents that are cytotoxic
during specific phases of the cell cycle (9). Appropriate timing
of administration of these agents should allow a high percent
age of the proliferating population to pass through the sensitive
phase of the cycle during periods when the drug concentration
in the blood is within cytotoxic levels.
The technic described here which utilizes a dispersed cell
preparation from solid tumors has yielded reproducible results
from studies of the cell cycle of S-180 and other tumors pres
ently under investigation. The possibility exists that the nuclei
in some stages of the cycle might be more fragile than in other
stages and, consequently, be totally disrupted during the pro
cedure. This would not be of concern in determination of the
cycle from appearance of the labeled mitotic cells unless the
mitotic figures were lost and even then, if the damage were
limited to a particular stage of mitosis, the loss would not
significantly affect the results. It would seem likely, in view
of the reproducibility of the results, that the dispersed cell
samples are representative of the entire cell population or that
cells in the same part of the cycle are lost in each preparation.
The technic has the advantage of yielding intact nuclei for
subsequent radioautographs rather than the fragmented nuclei
726
resulting from thin sections and lends itself well to use by
investigators not highly trained in histology.
ACKNOWLEDGMENTS
The authors wish to thank Miss Tommie Lou Barker and Mrs.
Carolyne M. Andrews for maintaining the tumor line and execut
ing the animal experiments and to gratefully acknowledge the
inspiration offered by Dr. Howard E. Skipper.
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CANCER
RESEARCH
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VOL. 28
The Mitotic Cycle of Sarcoma 180
Linda Simpson-Herren, Jane G. Blow and Paulette H. Brown
Cancer Res 1968;28:724-726.
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