[CANCER
RESEARCH 38, 809-814,
March 1978]
Induction of Concentration-dependent
Blockade in the G2 Phase of the
Cell Cycle by Cancer Chemotherapeutic Agents1
Bruce F. Kimler,2 Martin H. Schneiderman,3 and Dennis B. Leeper
Laboratory of Experimental
Philadelphia, Pennsylvania
Radiation
Oncology,
Department
of Radiation
Therapy and Nuclear
ABSTRACT
The mitotic cell selection procedure for cell cycle anal
ysis was utilized with Chinese hamster ovary fibroblasts
to determine the transition points in G,, i.e., the age in G,
at which cells become refractory to drug-induced progres
sion blockade, for several cancer Chemotherapeutic
agents and antimetabolites over a 1000-fold concentration
range.
The G transition points for five anticancer drugs (actinomycin D, Adriamycin, lucanthone, mitomycin C, and
bleomycin) varied linearly as a function of the logarithm
of the drug concentration between the S-G_,boundary at
low concentrations and prometaphase (45 min prior to the
end of karyokinesis) at high concentrations. Very low
concentrations produced an incomplete blockade with
some cells continuing to progress through the cycle.
Above a certain concentration the transition point did not
decrease further but attained a minimum value at 45 min
prior to the end of karyokinesis, implying that once a cell
has entered prometaphase it is completely refractory to
drug action and proceeds through mitosis without pertur
bation.
In contrast, the age at which cells are refractory to
treatment with three antimetabolites did not show a drug
concentration dependence. Over wide ranges of concen
tration, hydroxyurea, cycloheximide and puromycin re
sulted in transition points of 116 (the S-G boundary), 56,
and 58, min respectively, prior to the end of karyokinesis.
We interpret these results to indicate a physical mech
anism, distinct from pleotrophic action, by which the
anticancer antibiotics block the progression of G cells.
INTRODUCTION
Many of the cancer-chemotherapeutic agents currently in
clinical use share the property of inhibiting the progression
of cells through the growth cycle in addition to causing
cytotoxicity. If the cell cycle blockade induced by these
agents is reversible and blockade lasts only as long as the
intracellular titer of the drug is sufficiently high, then one
should be able to take advantage of the induced synchrony
in scheduling subsequent treatment. Although many re
ports have documented the effects of Chemotherapeutic
agents on cell cycle kinetics (1-5, 7-16, 19-31); most of the
techniques used lacked the necessary sensitivity required
' Supported by USPHS Grants CA 11602 and CA 16110.
2 Present address: Department of Radiation Oncology, University
sas Medical Center, Kansas City, Kans. 66103.
3 To whom requests for reprints should be addressed.
Received June 13, 1977; accepted December 8, 1977.
of Kan
Medicine,
Thomas Jefferson
University
Hospital,
for detailed analysis. Thus, results have been limited to
describing the location of a block in terms of general
phases of the cell cycle, i.e., G,, S, G,,, M, or at the
boundary between 2 phases. Even when greater precision
was attained, seldom was a concentration dependence of
the location of the block observed (29).
Using the mitotic cell selection procedure for cell cycle
analysis (26), we have determined the number of cells
refractory to drug-induced G-,blockade after treatment with
various concentrations of several cancer Chemotherapeutic
agents and antimetabolites. We were able to calculate the
time in G2 at which a particular concentration of drug
inhibited progression or induced a delay.
MATERIALS
AND METHODS
Cell and Culture Conditions. Chinese hamster ovary
CHO4 fibroblasts were maintained in exponential growth as
monolayer cultures in 75-sq cm Falcon plastic flasks at 37°
in a humidified atmosphere of 5% CO2in modified McCoy's
Medium 5A (Grand Island Biological Co., Grand Island, N.
Y.). The medium was supplemented with 10% fetal calf
serum (Flow Laboratories, Rockville, Md.) and antibiotics.
Stock cultures were maintained in antibiotic-free medium
and were found to be Mycoplasma free by the method of
Levine (18). Under these conditions the cells exhibited a
generation time of approximately 12 hr, with Gì= 3 hr, S
= 7 hr, G2 = 1.2 hr, and M = 0.7 hr.
Mitotic Selection Technique. The progression of cells
into mitosis was monitored by means of the mitotic cell
selection procedure for cell cycle analysis as described by
Schneiderman ef al. (26). The specific techniques utilized
have been published previously (16). Briefly, the rate at
which cells progress into a narrow selection window [22-4
min, with 16 min as the average, prior to the end of
karyokinesis (reformation of the nuclear membrane in the
daughter cells)] was determined by counting the number of
mitotic cells dislodged by a 20-sec selection shake repeated
every 10 min. Cell volume was routinely monitored by a
Coulter Channelizer to verify that the selected cells were
mitotic in size. In addition, the mitotic index was periodi
cally determined microscopically to verify that the selected
cells were 2:95% mitotic. If the mitotic index of the control,
scored microscopically, was not 95% or greater, an experi
ment was discarded. Prescott (23) has recently given the
name retroactive synchrony to this technique.
Drug Treatment. Hydroxyurea (NSC 32065), cyclohexi
mide (NSC 185), puromycin (NSC 3055), actinomycin D
(NSC 3053), lucanthone (NSC 14574), and mitomycin C
4 The abbreviation used is: CHO. Chinese hamster ovary.
MARCH 1978
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809
S. F. Kim 1er et al.
(NSC 26980) were obtained commercially from Calbiochem,
San Diego, Calif. Adriamycin (NSC 123127) was provided by
the Drug Development Branch, Division of Cancer Treat
ment, National Cancer Institute. Bleomycin (NSC 125066)
as Bleoxane was a gift from Bristol Laboratories, Syracuse,
N. Y. The drugs were dissolved in complete medium prior
to the start of an experiment. Treatment was accomplished
by replacing control medium with drug-containing medium
at the desired concentration.
Analysis of Data. When a drug that blocks G; progression
is added to an asynchronous population of cells, the yield
of mitotic cells (the number of cells selected at mitosis)
remains constant for a short period of time and then
declines to a level dependent upon drug concentration.
When the number of mitotic cells selected per shake is
plotted as a function of time after treatment, the resultant
curve can be resolved into 3 parts (e.g., utilizing actinomycin D; see Chart 1): (a) an upper plateau equal to the control
rate of selection; (b) a log-normal decline in the number of
selected cells; and (c) a lower plateau, less than 5% of the
control value, representing a background level of interphase cells and debris.
The mean time that a particular concentration of a drug
takes to prevent cell progression is estimated graphically
by determining the time from the initiation of treatment to
the point representing 50% of the control number of mitotic
cells. This time is subsequently referred to as the midpoint.
The transition point, operationally defined as the last point
in the cell cycle at which cells can be delayed by a particular
agent, is then estimated by adding the average time of
selection, i.e., 16 min prior to the end of karyokinesis, to
the midpoint.
In the charts the calculated value of the transition point
was plotted as a function of logarithm of the drug concen
tration (/¿g/mland ¿tM).Different symbols in the charts
represent different experiments. The straight line portions
of the curves in Charts 2 to 6 were fitted by linear regression
analysis, and the curved portion was fitted by eye.
To determine whether the effect of the drugs were due to
an alteration of the "selectability" of mitotic cells, we
analyzed the mitotic index after a 5-hr Colcemid accumula
tion in the presence of each drug. The results showed no
accumulation of mitotic cells. This indicates that the effect
on cell progression determined by mitotic selection was not
due to drug-induced changes of surface properties of the
cells. Although the results obtained with this technique do
not directly enable us to distinguish between drug concen
tration-dependent blockade occurring at various ages in G2
and drug concentration-dependent induction of blockade
as a function of 62 age but with the actual block at a single
point late in 62, small differences in G2 cell sensitivity are
quantifiable (25).
RESULTS
Actinomycin D. The effect of continuous exposure to
various concentrations of actinomycin D (1.0 to 125 /*g/ml)
on the selection of mitotic CHO cells is shown in Chart 1.
Mitotic cells continued to enter the selection window at the
control rate for a period of 20 to 60 min after exposure to
actinomycin D and then declined to a concentration-de810
292Concentration
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MINUTES
AFTERINITIATIONOf TREATHENT
Chart 1. Effect of various concentrations of actinomycin D on the rate of
selection of mitotic cells. At time 0, the control medium was replaced with
medium containing the indicated concentrations of actinomycin D for the
duration of the experiment. Exp , experiment.
pendent lower plateau. The midpoint the mitotic yield
curves decreased with increasing actinomycin D concentra
tion (up to 50 pig/ml). The increasing concentration also
resulted in a progressive shortening of the upper plateau
region. Concentrations of 1 to 5 ¿¿g/ml
did not completely
reduce the mitotic yield to 0. In this experiment the transi
tion point (midpoint plus 16 min) for G2 blockade by acti
nomycin D shifted from 106 min (1 ^g/ml) to 42 min (50 and
125 /¿g/ml,respectively) before the end of karyokinesis.
The G2 transition point as a function of the logarithm of
actinomycin D concentration is plotted in Chart 2. The
composite curve for actinomycin D (and the curves in
Charts 3 to 6 for the other drugs tested) was described by a
straight line as the transition point occurred closer to
mitosis with increasing drug concentration until a plateau
region was attained where the transition point no longer
decreased with increasing concentration. The linear portion
of the curve was calculated over a concentration range
from 0.8 (transition point corresponding to the S-G2bound
ary) to 40 /tig/ml (transition point corresponding to prometaphase) and exhibited a slope constant (m) of 46 (see
Table 1). For concentrations of actinomycin D greater than
40 pig/ml, the transition point remained at 42 ±1 min prior
to the end of karyokinesis. For comparison, Dewey and
Highfield (8, 12), using the same CHO cell line and tech
nique, reported G2transition points of 120, 79, and 52 min
prior to the end of karyokinesis for 2, 5, and 15 /*g
actinomycin D per ml, respectively. These data (8, 12)
correlated well with Chart 2.
Adriamycin. A response similar to that obtained with
CANCER
RESEARCH
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VOL. 38
Concentration-dependent
CONCENTRATION, »M
10
100
uo
G2 Transition Points
CONCENTRATKDN,
1000
100
K>
Actmomycin-D
WOO
Adriamycin
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120
. 100
100
80
80
60
40
40
0.1
1
10
100
1000
0.1
1
CONCENTRATION, pg/ml
Chart 2. Effect of actinomycin D concentration on the transition point
(i.e., the time prior to karyokinesis, after which cells are refractory to drug
treatment and proceed through mitosis). •,•different experiments.
no
10
1000
CONCENTRATION, vg/mi
Chart 3. Effect of Adriamycin concentration on the transition point. The
different symbols refer to different experiments; A, •.•continuous
exposure to Adriamycin; A, O pulsed exposure of 10 to 30 min.
Table 1
Parameters of concentration-dependent G2transition point
regression line
T = m log C/Co + T0°
CONCENTRATION, uM
10
1000
ICO
140
DrugActinomycin
tion
range1-500.3-501-200.1-3001-505-1000.4-40m46254225416836C^g/rnl504820*500MM429550T»
(min)41454547 £ 120
DAdriamycinLucanthoneMitomycin
5
_. roo
CBleomycin0Concentra
" T, G2transition point (min) before completion of karyokinesis
produced by a drug concentration (C); m, slope constant; C0,
minimum drug concentration yielding the plateau transition point,
T"A
Solubility limitation of lucanthone, 20 pg/ml.
c Three separate lots of bleomycin; see Chart 6.
actinomycin D was observed with Adriamycin. As shown in
Chart 3, the location of the Adriamycin G2 transition point
was also dependent upon drug concentration. There is a
linear relationship between the transition point and the
logarithm of the concentration of adriamycin between 0.3
and 50 /u.g/ml. The transition point decreased from =105
min (S-G2boundary) at 0.3 /ug/ml to 45 min (prometaphase)
at 50 /^g/ml with a slope constant (m) of 25 (see Table 1).
Concentrations of Adriamycin greater than 50 /ng/ml did
not further alter the transition point, which was located 45
±3 min prior to the end of karyokinesis. Results of treat
ments pulsed for 10 to 30 min were similar to continuous
treatment.
Lucanthone. Lucathone is a thiaxanthenone analog of
actinomycin D and Adriamycin that intercalates into DNA
and inhibits RNA and DNA synthesis and also inhibits the
accumulation and repair of sublethal radiation damage (6,
17). Unlike actinomycin D and Adriamycin, which bind
irreversibly to DNA, lucanthone only loosely binds to DNA,
and consequently its effects are completely reversible (6,
17). However, the relationship between lucanthone concen-
z
m
i"
60
40
OJ
1
10
100
1000
CONCENTRATION, pg/ml
Chart 4. Effect of lucanthone concentration on the transition point. The
different symbols refer to different experiments; •,•continuous exposure
to lucanthone; D, O, pulsed exposure of 60 to 120 min.
tration and the G2transition point (Chart 4) is similar to the
other intercalating antibiotics. Although it was not possible
to test concentrations of lucanthone greater than 20 ¿ig/ml
because of solubility limitations, the results of an experi
ment (not shown) with a slurry of 50 ¿tg/mlindicated that a
constant mimimum value of the transition point existed at
45 min prior to the end of karyokinesis. The slope constant
of the transition point plotted as a function of logarithm of
lucanthone concentration was 42 (Table 1).
Mitomycin C. The dependence of the G2 transition point
on mitomycin C concentration is shown in Chart 5. Again,
because of solubility limitations, it was impossible to
achieve high enough concentrations of the drug to demon
strate a minimum plateau for the transition point. However,
the transition point varied with concentration of mitomycin
C over the range of 1 to 300 ¿¿g/ml
and exhibited a slope
constant of 25 (Table 1).
Bleomycin. The relationship between the G2 transition
MARCH 1978
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
811
B. F. Kim 1er et al.
CONCENTRATION, uM
10
100
woo
140
â„¢ 120
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K»
*
80
g
60
40
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1
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CONCENTRATION,
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CONCENTRATION . ug/ml
Chart 6. Effect of bleomycin concentration on the transition point. •.•
iriments with 3 different lots of bleomycin
bleomvcin
Chart 5. Effect of mltomycin C concentration on the transition point. •, A, 3 experiments
•different experiments.
point and the concentration of bleomycin for 3 separate lots
of bleomycin is shown in Chart 6. All 3 curves shown in
Chart 6 exhibit a concentration dependence of the G-,
transition point, but the slope constants of the curves vary
by a factor of 2 (Table 1). Although 1 lot of bleomycin was
suspect because it was out of date and had been stored
under unknown conditions (curves composed of solid tri
angles), the other 2 lots, gifts directly from Bristol Labora
tories, were also outdated but were guaranteed to have full
clinical potency. Considering the number of constituents in
bleomycin, it is not surprising that a difference in the
concentration dependence of the G2transition point occurs
between lots. The minimum value of the transition point
induced by the highest concentration of bleomycin was 47
min prior to the end of karyokinesis, and the slope con
stants for the 3 lots were 36, 41, and 68 (Table 1).
Hydroxyurea. When cells were exposed to either 1.0 or
10 IHM hydroxyurea (Chart 7), the rate at which mitotic cells
entered the selection window remained equal to the control
for =20 min and then declined to 0 at about 220 min after
initiation of treatment. The transition point was at 116 ±3
min prior to the end of karyokinesis (average of 19 experi
ments). After a 10-min pulse of tritiated thymidine to label S
phase cells, the number of selected unlabeled mitotic cells
(i.e., cells in G, or M during the 10-min pulse) was equal to
the number of cells refractory to hydroxyurea treatment. In
addition, no radioactivity was detected in cells selected
when hydroxyurea was added coincident with tritiated thy
midine. These results indicate that the location of hydrox
yurea transition point is at the S-G. boundary. At a concen
tration of 0.1 rriM hydroxyurea there was incomplete block
ade of cells, and a transition point determination could not
be accurately made (26).
Cycloheximide. Treatment with cycloheximide at con
centrations between 0.1 and 100 /ug/ml completely sup
pressed the progression of mitotic cells into the selection
window with a transition point of 56 ±1 min prior to the
end of karyokinesis (Chart 7). For each concentration, the
mitotic yield was equal to the control for approximately 30
min and then declined to 0 over the next 50 min.
Puromycin. At concentrations above 50 ¿¿g/ml,
this pro-
812
•
Cycloheximide
140
Puromycin
. Hydroxyurea
120
80
60
40
0.1
wo
l
CONCENTRATION,
1000
ug/ml
Chart 7. Effect of cycloheximide. puromycin. and hydroxyurea concen
tration on the transition point. The G2 transition point is independent of
concentration with these antimetabolites. Cycloheximide data from 6 exper
iments, puromycin data from 3 experiments, and hydroxyurea data from 6
experiments.
tein-synthesis inhibitor produced complete blockade of G-,
progression with a constant transition point of 58 ±1 min
(Chart 7). At lower concentrations there was incomplete
suppression, but cells that were blocked exhibited the
same transition point. The puromycin and cycloheximide
transition points were indistinguishable.
DISCUSSION
By monitoring the rate at which mitotic cells were selec
tively detached from an asynchronously growing monolayer, we were able to determine the point in the cell cycle
(the transition point) at which various concentrations of
DNA-active agents and antimetabolites act to interfere with
the progression of cells into mitosis.
The antimetabolic drugs, hydroxyurea, an inhibitor of
DNA synthesis, and cycloheximide and puromycin, inhibi
tors of protein synthesis, have transition points represent
ing the last point in the cell cycle when synthesis of an
essential precursor for progression occurs (19). Since cyCANCER
RESEARCH
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VOL. 38
Concentration-dependent
cloheximide and puromycin inhibit protein synthesis, any
cell in which the last protein(s) essential for mitosis had
been synthesized would continue through division without
delay in the presence of these drugs, while a cell that had
not synthesized that protein (i.e., any cell earlier in the cell
cycle than =57 min prior to the end of karyokinesis) would
be unable to progress further. Agents that act by such a
mechanism are characterized by single, concentration-in
dependent transition points (Chart 7).
CHO cells treated with intercalating antibiotics (actinomycin D, Adriamycin, and lucanthone) and the DNA-active
agents (bleomycin and mitomycin C) displayed concentra
tion-dependent G2transition points. At low concentrations,
the transition point was at or near the S-G2 boundary. Over
the intermediate-to-high concentration ranges, there was
complete suppression of cell progression in G2.The number
of cells refractory to the drug decreased with increasing
concentrations, and the transition points for the high drug
concentrations occurred later in G2. The slope constant (m
in Table 1) of the regression line produced when the
transition point is plotted against the logarithm of drug
concentration is a measure of the relative effectiveness of a
particular drug in limiting the number of refractory cells.
Thus, actinomycin D (m = 46) is more effective on a per
molecular basis than lucanthone (m = 42), which in turn is
more effective than Adriamycin (m = 25) or mitomycin C (m
= 25). The slope constants for 3 lots of bleomycin ranged
from 36 to 68, indicating a nearly 2-fold difference in
efficacy between lots of bleomycin that were stated to
possess full clinical potency.
For the intercalating antibiotics and 1 lot of bleomycin,
there was a constant minimum transition point (7,,) once a
certain drug concentration (Cn) had been exceeded (see
Table 1). Because of insufficient information concerning
uptake rates and the intracellular concentrations that might
be reached for these drugs, it is not possible to use d, as a
definitive parameter. However, the constant value of Tufor
each drug, with a narrow range between 41 and 47 min
prior to the completion of karyokinesis, indicates a common
temporal event such that, once a cell has progressed
beyond it, further progression through mitosis cannot be
prevented, regardless of drug concentrations.
Our results indicate at least 2 different mechanisms by
which drugs block the progression of mammalian cells into
mitosis. The first mechanism is inhibition of the synthesis
of the molecule(s) essential for division and is characterized
by transition points for drug-induced division blockade that
are concentration independent. The antimetabolites fall
into this category. The second mechanism, physical pertur
bation of chromatin structure or division apparatus, is
characterized by concentration-dependent transition points
for drug-induced division blockade. The DNA-active drugs
fall into this category.
Although other investigators have observed that Adria
mycin causes a G2 block with subsequent accumulation of
cells in G2(1-4,14,16, 28), with the exception of a previous
detailed report from this laboratory (15), the only observa
tion that the transition point was dependent upon Adriamy
cin concentration was reported by Clarkson and Humphrey
(7). Similarly, it has been shown that actinomycin D causes
a G, progression blockade (8, 9, 11, 12, 22, 24, 30) and
MARCH
G2 Transition Points
Dewey and Highfield (8,12), also using the mitotic selection
procedure, were able to discern different transition points
for 3 concentrations of the drug.
Epifanova ef a/. (10) observed a time- and concentrationdependent decrease in the mitotic index of CHO cells after
the addition of lucanthone; however, they could not differ
entiate G2transition points. Nowell (21) was able to show a
similar decrease in the mitotic index after treatment with
mitomycin C, but again, differentiation of the transition
points could not be observed. Several investigators have
reported that bleomycin causes a G2block and an accumu
lation of cells in G2 (5, 13, 20, 27). Although different
positions for the G2 block were reported, no concentration
effect was observed. Our data showing the lot-to-lot varia
tion in the efficacy of bleomycin should be taken as a
warning both to the research and clinical communities of
the possible heterogeneity of different preparations for
bleomycin.
Finally, we have shown that for the intercalating antibiot
ics and for bleomycin there exists a common minimum
transition point at approximately 45 min prior to the end of
karyokinesis that cannot be altered by high concentrations
of drug. The commonality of the minimum transition point
at high concentrations suggests that at this point in the cell
cycle (probably prometaphase)5 the cell is refractory to
drug-induced blockade. Since the transition point for inhi
bition of DMA synthesis (hydroxyurea transition point) or
inhibition of protein synthesis (cycloheximide or puromycin
transition point) is concentration independent, it is unlikely
that the anticancer drugs studied block cell progression
only by inhibition of macromolecular synthesis. Therefore,
it seems reasonable that the intercalating antibiotics, by
binding to DNA, or mitomycin C, by cross-linking DNA, or
bleomycin, by causing DNA strand breaks, induce concen
tration-dependent structural defects in DNA that interfere
with either chromatin condensation, spindle fiber forma
tion, or attachment of spindle fibers to the centromere or
centriole and thus prevent normal progression of G2 cells
towards or into mitosis.
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S. F. Kimler et al.
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CANCER
RESEARCH
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VOL. 38
Induction of Concentration-dependent Blockade in the G2
Phase of the Cell Cycle by Cancer Chemotherapeutic Agents
Bruce F. Kimler, Martin H. Schneiderman and Dennis B. Leeper
Cancer Res 1978;38:809-814.
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