[CANCER RESEARCH 33,691-694,
Aril 1973]
Response of Mammalian Cells following Treatment with
Bleomycin and l,3-Bis(2-chloroethyl)-l-nitrosourea during
Plateau Phase1
S. C. Barranco, Judy K. Novak, and Ronald M. Humphrey
Departments of Surgery fS. C. B., J. K. N.J and Physics, [R. M. H.J, The University of Texas at Houston M. D. Anderson Hospital and Tumor
Institute, Houston, Texas 77025
SUMMARY
One hr of exposure to bleomycin (100 jug/ml) during the
plateau phase caused a 10-fold greater decrease in the survival
fraction of Chinese hamster ovary cells than was caused by
bleomycin treatment during the exponential growth period.
One hr of exposure to l,3-bis(2-chloroethyl)-l-nitrosourea
(25
Mg/ml) had no effect on the survival fraction of cells treated in
the exponential growth period; however, the same dose
reduced the survival fraction of cells treated in plateau phase
to 0.0002 (this represents a 500-fold decrease in the survival
fraction).
INTRODUCTION
Immediately after mammalian cells are subcultured in vitro,
a lag period of growth is observed, followed by a period of
exponential growth. If the incubation time is extended, the
growth rate slows so that a plateau phase is ultimately
achieved. The onset of plateau phase is presumably caused by
the exhaustion of one or more essential nutrients from the
medium (8) or by contact inhibition (1). Froese (6) reported
the existence of a high-molecular-weight component in "used
medium" that inhibits growth of exponentially growing cells.
Autoradiographic studies by Tobey and Ley (15) showed
that CHO2 cells in stationary phase did not incorporate
TdR3H into DNA. Microfluorometric measurements snowed
that the cells contained a G! complement of DNA and, upon
resuspension of the plateau cells at lower numbers in fresh
medium, DNA synthesis always preceded cell division.
Furthermore, they were able to show (16) that plateau phase
could be induced merely by growing cells in isoleucine-deficient medium for 24 to 36 hr (or about 2.5 cell cycle times).
The addition of isoleucine to the plateau phase culture causes
the cell population to initiate DNA synthesis; subsequently,
the cells divide. These data are evidence that cells in plateau
phase are arrested in the Gt period. The G0 compartments
described by Patt and Quastler (13) for stem cells and by
Mendelsohn (11, 12) for tumor cells in the viable but
1This work was supported by American Cancer Society Institutional
Research Grant ACS-IN-43-L.
JThe abbreviations used are: CHO, Chinese hamster ovary; TdR-3H,
thymidine-methyl-3H; BLM, bleomycin; BCNU, 1,3-bis(2-chlorocthyl)1-nitrosourea.
Received September 8, 1972; accepted December 28, 1972.
nongrowth fraction of some solid tumors thus resemble
plateau phase cells. For this reason, plateau phase cultures may
serve as an excellent model system for studies of the ability of
chemotherapy drugs to induce damage or death in populations
of mammalian cells maintained in a viable but nondividing
state during treatment. This paper reports the survival
responses of mammalian cells in plateau phase to BLM and
BCNU.
MATERIALS
AND METHODS
Cell and Culture Techniques. The CHO cell line stocks were
maintained in monolayer cultures in McCoy's 5A medium
supplemented with 20% fetal calf serum in a 5% CO2,
humidified incubator at 37°.The average cell cycle time under
these conditions is 13 hr with a 2.5-hr pre-DNA synthesis
period (G,), an 8-hr DNA synthesis phase (S), and a 1.5-hr
post-DNA synthesis period (G2). The population-doubling
time (TD) was 14 hr, and the plating efficiency ranged from
80 to 95% during the experiments.
Induction of Plateau Phase. Plateau phase was achieved by
pipetting a known number of exponentially growing cells
(between 2 and 3 X IO6 cells, as determined on a Coulter
counter) into 60-mm plastic Petri dishes containing 5 ml of
McCoy's 5A medium, supplemented with 20% fetal calf serum.
We monitored the increase in cell number by obtaining total
cell counts on replicate plates of cells at various times
thereafter. We found that, whenever more than 2 X IO6 cells
are placed in 60-mm Petri dishes, the TD becomes 20 to 22 hr.
The onset of plateau phase was characterized by the further
slowing of the rate of cell division. Once plateau phase was
reached (about 6 to 8 X 10" cells/Petri dish), no net increase
in cell number was observed. In our plateau (and in those used
in other laboratories), a small but measurable fraction (1 to
4%) of cells continues to incorporate TdR3H throughout the
incubation. Presumably, these cells are still able to progress
around the cell cycle, but at a slower than normal rate.
Meanwhile, more than 95% of the cells in the experimental
population are delayed in GÌ
phase.
Cell Progression Studies on Cells Reentering the Cell
Division Cycle from Plateau Phase. When plateau phase cells
are resuspended at lower cell concentrations in fresh medium,
they reenter the cell cycle in a synchronous wave, and DNA
synthesis always precedes mitosis. This progression was
APRIL 1973
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691
S. C. Barranco, J. K. Novak, and R. M. Humphrey
monitored in the following manner. Plateau phase cells (IO6)
in 5 ml of fresh McCoy's 5A medium were plated into each
60-mm Petri plate and incubated at 37°.Each hr thereafter
(for 36 hr), repÃ-cateplates of cells were pulse labeled for 10
min with TdR-3H, 1 ¿uCi/ml,1.9 Ci/mmole (Schwarz/Mann,
Orangeburg, N. Y.). The pulse labeling was accomplished by
the addition of enough TdR-3H directly to the medium in
which the cells were growing to achieve a final concentration
of 1 //Ci/ml. The cells were reincubated for 10 min at 37°in a
COj incubator. Following the pulse labeling, the cells were
washed with fresh McCoy's 5A medium and were removed
from the plates by trypsinization (0.025% for 5 min). The cells
were then centrifuged, the pellet was fixed in 50% glacial
acetic acid and stained with 2% aceto-orcein, and squash
preparations of the cells were made on glass slides.
In all experiments involving the assay of populations of
radioactive labeled cells, liquid emulsion (Ilford K5) autoradiography techniques were used. The autoradiography slides
were maintained in a lightproof box at 4°for 10 days and
were developed in Kodak D19 developer. The movement of
cells into S phase was reflected by an increase in the number
of labeled cells with time.
Survival Determinations on Plateau Phase Cells. The effects
of drugs on cell survival were determined with cells that had
been maintained in plateau phase for at least 24 hr.
Concentrations of from 1 to 100 ¿tgof BLM or BCNU per ml
were used in these studies. We treated the cells by adding the
drug directly to the medium in which the cells had been
growing, since the cells might have progressed out of plateau
phase and back into the cell cycle during the treatment if drug
diluted in fresh medium had been used. Eight replicate plates
of plateau cells were used in each survival study. Seven plates
received different concentrations of drug, and the 8th plate of
cells served as the control. The cells were treated in a CO2
incubator at 37°for 1 hr. In all cases in which the cytotoxic
action of the drugs was tested, survival was determined by the
ability of the treated cells to form colonies. After the cells
were treated, the medium was decanted and the plates were
washed twice with fresh medium. The cells were removed from
the plates by trypsinization (0.025%) and were counted.
Known numbers of single cells were plated into plastic Petri
dishes and incubated in a CO2 incubator for 6 to 8 days at
37°.Colonies were fixed in methanol, stained with 4% crystal
violet, and counted. A cell was considered to have retained
reproductive capacity (viability) if it gave rise to a colony of
50 or more cells. Each survival determination was repeated 3
times.
Drug Solutions. The drug solutions always were prepared
immediately before use to ensure against loss of activity. BLM
was dissolved in 0.85% sodium chloride solution, and BCNU
was dissolved in absolute ethanol. Both drugs were then
diluted to the final treatment concentrations in fresh McCoy's
5A medium. The pH of the treatment
between 7.2 and 7.4.
good viability (85% plating efficiencies) through 50 hr in
plateau phase. Survival determinations were made on cells that
had been in plateau phase for at least 24 hr. At the same time,
other plateau phase cells were counted and plated into fresh
McCoy's 5A medium. Cell counts and studies of TdR-3H
incorporation were done to enable us to determine whether
plateau phase cells were able to reenter the cell cycle upon
return to fresh medium. The fraction of cells incorporating
TdR-3H (S phase cells) or the fraction of cells dividing were
calculated with (N/N0 ) —¿
1 [where -/V0= the total cell number
or the number of TdR-3H-labeled cells at the time that the
cells were resuspended in fresh medium (0 hr), and N = the
total cell number or number of TdR-3H-labeled cells at each
particular sample time].
Chart 1 shows that, upon resuspension at lower cell
numbers in fresh medium, the plateau phase cells recover and
move synchronously through CM and into S phase within 12
hr and approximately double in number by 30 hr. As the cells
progress from G] into S phase, the labeling index rises,
accounting for the increased fraction of cells in S phase seen in
Chart 1. The cells that had been in plateau phase in our system
take about 5 times longer to reach S phase after they were
resuspended in fresh medium than did cells obtained by
mitotic selection (2, 3, 14). We can offer no explanation at
this time for the difference in our data and those of Tobey and
Ley (15), who have shown that the CHO cells in their system
enter S phase 4 hr after resuspension and divide after 12 hr.
This phenomenon
is currently being investigated in our
laboratory.
Although Hahn (8) has shown that RNA and protein
synthesis continue in plateau phase cells, before the cells can
progress back into the cell cycle it is likely that they must
transcribe new mRNA, and "resynthesize" enzymes essential
for the initiation of DNA synthesis. Patt and Quastler (13)
have shown that in vivo G0 cells reenter the cell cycle through
the G, phase, and the data of Chart 1 indicate that the plateau
phase cells in our system behave in a similar manner.
The effects of BLM on survival of cells treated in plateau
phase is shown in Chart 2. Survival curves of asynchronous
cells and of G, phase cells are included for comparison. (These
2 curves were published previously, and the methods for
obtaining them may be found in Ref. 3.) All cells were treated
in their respective phases and plated immediately for colony
1.0
Fraction
o /
dividing
/
0.5
medium was always
RESULTS AND DISCUSSION
By varying the initial cell number, plateau phase can be
attained in about 48 hr. The monolayer of cells maintained
„¿a,'
B g
0 2
692
Fraction
in S phase /
Resuspend
Cells
IO
20
30
Hours
Chart 1. The fraction of cells entering S phase (incorporating
TdR-3H) or the fraction of cells dividing following the resuspension of
plateau phase cells in fresh medium.
CANCER
RESEARCH
VOL.
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33
Cell Response to BLM and BCNU during Plateau Phase
formation. A previous study (3) showed that BLM kills cells
specifically in the M and G2 phases of the cell cycle.
All 3 curves in Chart 2 are biphasic, and the absence of a
shoulder region on the curves suggests either that the cells do
not accumulate any sublethal damage (4, 5) or that the
smallest amount of damage induced by BLM is enough to
cause lethality. Cells treated in plateau phase with 100 ¿/gBLM
per ml are 10 times more sensitive than those treated in the
exponential growth period and are about 30 times more
sensitive than synchronized GÌ
cells. This finding suggests that
plateau phase cells may have a decreased capacity for the
repair of damage caused by BLM. This possibility is being
investigated in our laboratory.
The effects of treatment with an "alkylating agent" are
shown in Chart 3, which shows that plateau phase cells were
extremely sensitive to BCNU. A dose of 25 ¿igBCNU per ml
for 1 hr reduced the survival fraction of plateau phase cells to
0.0002 but had no effect on an asynchronous population.
(The curve for the asynchronous population was published
previously and the methods by which it was obtained can be
found in Ref. 2.) BCNU kills cells in all phases of the cell
cycle, cells at the G! -S boundary and in mid-S phase being the
most sensitive (2).
The very small shoulder region of the plateau phase BCNU
survival curve (compared with that of cells treated in
exponential growth) indicates a greatly reduced potential for
repair and recovery from sublethal damage (4, 5). It was
shown (7, 8) that, in X-irradiated plateau phase cultures, cell
survival may be enhanced if, before the cells are subcultured
for colony formation, the culture is left in plateau phase for
several more hr. In addition, Little (9, 10) has reported the
presence of a diffusible substance released by plateau phase
cells which may promote repair of sublethal damage in plateau
Synchronized
G,cells
B O.I
s
\
Asynchronous
log growing cells
01
£ 001
Plateau
phasecells
0001
00001
10
30
50
70
100
Dose BCNU(jjg/ml) for Ihr
Chart 3. The effects of BCNU on survival of CHO cells treated with
BCNU in the exponential phase of growth or in plateau phase.
phase and in exponentially growing cells. It is possible that, by
holding these drug-treated cells in plateau phase conditions for
longer periods after treatment and then plating them for
colony formation, survival may be enhanced. This possiblity is
currently being investigated in our laboratory.
The apparently high killing efficiency of BLM and BCNU on
plateau phase cells in vitro cannot necessarily cause one to
infer that these agents will be effective agents for treating G0
cells. We are aware of the limitations inherent in comparing
plateau phase cells with G0 cells in vivo. However, these results
do indicate that the drugs are much more effective for a given
concentration on plateau phase cells than on cycling cells.
Hence, there is a possibility that considerable killing in the G0
compartment in vivo may be observed. A better knowledge of
the effects of anticancer drugs on these viable but nondividing
cell populations has potential clinical significance. Since
plateau or G0 cells generally are not killed by the cell
cycle-specific drugs, these cells probably contribute to the
repopulation or regrowth of the tumor. Therefore, it is
extremely important to identify and characterize drugs that
will specifically influence survival, repair, and repopulation
kinetics of plateau phase cells.
REFERENCES
0.01
10
30
50
70
100
Dose Bleo(jig/ml) for I hr
Chart 2. The effects of BLM (Bleo) on survival of CHO cells treated
while in the exponential phase of growth, or while in G, or plateau
phase.
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CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
Response of Mammalian Cells following Treatment with
Bleomycin and 1,3-Bis(2-chloroethyl)-1-nitrosourea during
Plateau Phase
S. C. Barranco, Judy K. Novak and Ronald M. Humphrey
Cancer Res 1973;33:691-694.
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