[CANCER
RESEARCH
34, 878-881, April 1974]
Effects of Bleomycin on Progression through the Cell Cycle
of Mouse L-Cells
Michinori
Watanabe,
Yosinobu Takabe, Takesi Katsumata,
and Toyozo Terasima
First Department of Medicine, Chiba University School of Medicine, Inohana, Chiba, Japan [M. W., Y. T., T. K.], and Division ofPhysiology
Pathology, National Institute ofRadiological Sciences, Anagawa-4, Chiba, Japan Er. T.]
MATERIALS AND METHODS
SUMMARY
The effect of bleomycin
on progression
of mouse L-cells
through the cell cycle was studied. In the presenceof bleo
mycin, the beginning of DNA-synthetic
at the same
and
rate
as that
(5) phase occurred
of the untreated
control,
but the
duration was prolonged. In cells treated pulse-wise with
bleomycin at the i phase, the prolongation of S phase was
slight, whereasthe G2 phaseshoweda considerabledelay.
Bleomycin also affected the entry of asynchronous
cells into
Cell Line. A clonal derivative of mouse L-cells (B929L2J), designated as the L5 strain, was used throughout
the experiments. The cells were grown as a monolayer in
F1OHI medium (3) with 5% calf serum added. The median
generation time was about 18 hr with a 5-hr G1 period, 9-hr
S period, and 3-hr G2 period.
Synchronous Culture of L-Cells. Synchronous cells were
obtained by a selection of mitotic cells from randomly
growing L-cells (14). The culture was initiated with I x l0@
mitosis by arresting them at the early half of the G2 phase. cells/ l80-ml square culture bottle. After 36 hr of incuba
Interference did not occur with transition of mitosis to the
tion, the medium was discarded to remove fragile and dy
G1 phase.
ing cells. The culture, fed with the prewarmed fresh
The observed prolongation of DNA-synthetic
phase in
the presence of bleomycin might be due to the inhibition of
DNA synthesis or the damage to DNA molecules by the
drug.
INTRODUCTION
Bleomycin is a glycopeptide antitumor antibiotic that
has been isolated from the culture filtrates of Streptomyces
verticillus (19, 20). In clinical studies, the drug has demon
strated certain characteristics different from those of other
chemotherapeutic
agents. Bleomycin shows selective ac
tivity on squamous cell carcinoma (2, 12) and malignant
lymphomas (5, 22); moreover, it is rarely accompaniedby
hematopoietic disturbance (2, 5, 12, 22), a major side ef
fect of other cytotoxic antitumon agents.
@
Studies on the mechanism of action of bleomycin at the
cellular level revealed that the drug affects most strongly
the synthesis of DNA and, to a lesser extent, protein and
RNA syntheses (11, 21). The drug also inhibits the prolif
eration of various mammalian cells in culture (I, 16). The
growth medium, was incubated for another 5 to 6 hr. Then,
the mitoses that appeared were readily shaken off by man
ual agitation of the bottle. The mitotic frequency of han
vested cells was usually over 95%. The harvested cells were
all attached to the bottom of plastic petni dishes (60 x 15
mm; Falcon Plastics, Los Angeles, Calif.) within 2 hr of
incubation in a CO2 chamber at 37°.The synchrony pro
cedure was carried out at 37°.
Determination of Mitotic Frequency. The cells growing
in plastic
dishes
were dispersed
with
0. 1% trypsin
( 1 : 250;
Difco, Detroit, Mich.) : saline D2. The cells were then cen
trifuged, fixed in acetic acid :ethanol (I : 3), and stained
with acetic orcein. The mitotic frequency for each sample
was determined by scoring 1000 cells.
Autoradiography
of Cells. Mitotic cells were inoculated
into plastic dishes in which coverslips were placed, and in
cubated in a CO2 chamber at 37°.At 3 hr, when cells were
attached on coverslips and the whole population of syn
chronous cells was in
phase of the cell cycle, bleomycin
was added to cultures to a final concentration
of 100
@g/ml. Thereafter, covenslips were taken from dishes at
particular biphasic survival curve of mouse L-cells was intervals of 2 hr and placed into medium containing thymi
reported previously and the implication was discussed by
us (13, 15). On the other hand, little information is avail
able regarding the effect of bleomycin on the cell cycle. Ac
cordingly, the present studies on bleomycin action were fo
cused on the progression of mouse L-cells. The findings
obtained
would be useful for the rational designing of can
cer chemotherapy.
Received September 7, 1973; accepted January 4, 1974.
878
dine-3H, 0.5 j@Ci/ml (5 Ci/mmole, Radiochemical Centre,
Amersham, England) for 20 mm. Then, cells were fixed in
acetic acid :ethanol (1 : 3), dried, and the autoradiographs
were prepared. For autoradiography,
the nuclear emulsion
(NRM-2, Konishiroku Photoindustry
Co., Tokyo, Japan)
was
applied
and
photographic
processes
were
followed.
The fraction of labeled cells was determined by scoring
1000 cells for each sample.
Colcemid Treatment of Cells. An exponentially growing
CANCER RESEARCH
VOL. 34
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Effects of Bleomycin
culture of L-cells was treated continuously with Colcemid,
0. 1 @tg/ml, and bleomycin, 100 zg/ml. In control culture,
medium contained Colcemid but no bleomycin. After the
addition of drugs, samples were taken at 30-mm intervals
over a 5-hr period. The entry of cells into mitosis was ex
amined by scoring the number of cells in mitosis in each
sample.
Bleomycin. Bleomycin AS (Lots 00702 and 6), a copper
free sulfate,
was obtained
from
Nippon
Kayaku
Co.
in
in
0
z
-J
Ui
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I-
z
Ui
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Ltd.,
Ui
Q.
Tokyo, Japan. The drug was dissolved in FlO medium and
kept in a refrigerator until used.
@
on Cell Cycle Progression
HOURS AFTER MITOSIS
Chart 1. Effect of bleomycin on progression of mitotic cells into
phase. The harvested mitotic cells were exposed continuously to bleomy
RESULTS
cm,
100
@ig/ml. At
30-mm
intervals,
samples
were
taken
and prepared
for
determination of mitotic frequency. For each sample, mitotic frequency
The effect of bleomycin on the progression of mitotic
cells into G1 phase is shown in Chart 1. For treatment,
cells in mitosis were suspended in medium containing bleo
mycin, 100 zg/ml, immediately after harvest and were
kept incubating at 37°.Samples were taken at 30-mm in
tervals,
and cells were dispersed
with 0. 1% trypsin.
ples taken during the 1st 2 hr of incubation,
floating
@
in culture
medium
were collected
tion of DNA-synthetic
In contrast,
phase was blocked
the comple
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12
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16
20
HOURS AFTER MITOSIS
Chart 2. Effect of bleomycin on the DNA-synthetic phase of syn
chronous L-cells. At 3 hr after mitosis, bleomycin was added into culture
medium to a final concentration of 100 @g/ml.Then, samples were taken
at intervals and pulse labeled with thymidine-'H (0.5 tCi/ml; 5 Ci!
mmole) for 20 mm. For each autoradiographic sample, the fraction of
labeled cells was determined by scoring 1000 cells.
In
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Ui
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by the drug.
The labeled fraction of the control culture dropped to 30%
@
Ui
and put together
of DNA-synthetic
cells was determined by pulse-labeling
cells with thymidine-3H at 2-hr intervals (Chart 2). The
beginning of DNA-synthetic
phase was not affected in
cells treated with bleomycin, since the labeled fraction of
both control and drug-treated cells increased at the same
rate from 5 hr onward and finally reached 90% at 9 hr af
of S phase).
0 Control
-J
-J
In sam
cells, bleomycin was added to culture medium to a final
concentration of 100 .tg/ml, and thereafter the fraction
(middle
100
U)
the cells still
with trypsinized cells. Then, cells were centrifuged, fixed,
and stained for determination
of mitotic frequency. The
harvested cells exposed continuously to bleomycin, 100
j@g/ml, underwent the same rate of decrease in mitotic
frequency as did untreated control cells. The values of
control and drug-treated cells dropped to 0% by I hr of
incubation, indicating that bleomycin did not affect the
transition from mitosis to G1 phase.
In order to examine the effect of the drug on the DNA
synthetic phase, autoradiographic
studies were performed
on synchronous L-cells. Three hr after harvest of mitotic
ter mitosis
was determined by scoring 1000 cells.
LI
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z
I-
Ui
at 15 hr, while 70% of drug-treated cells still remained in
S phase even at 17 hr. This indicates that bleomycin pre
vented the passage of cells from S to 2 phase. The dura
tion of S phase measured at the 50% level was, therefore,
prolonged 5 hr by the drug treatment. Incidentally, the
number of grains per labeled cells was apparently lower
in bleomycin-treated
cells than in untreated control cells.
Chart 3 shows the effect of pulse treatment on the cell
cycle progression. Three hr after mitosis, synchronous G1
populations were treated for 1 hr with bleomycin, 100
@g/ml. Then cultures were rinsed twice with FlO medium
and fed with fresh growth medium for further incubation.
The progression of G 1-treated cells through the following
HOURS AFTER MITOSIS
Chart 3. Effect of bleomycin on progression of synchronous cells pulse
treated at
phase. At 3 hr, bleomycin was added to cultures to a final
concentration of 100 @zg/ml.After 1 hr of incubation with the antibiotic,
the cultures were rinsed twice with FlO medium and fed with fresh FlO
medium for further incubation. After the treatment, samples were pulse
labeled with thymidine-3H at intervals and prepared for autoradiography
(circles). The division of synchronous cells was determined by repeated
cell cycle phases was examined by pulse-labeling cells with
scoring of cell number in specifiedmicroscopicfields (triangles).
z
Ui
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>
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Ui
Ui
0.
APRIL 1974
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1974 American Association for Cancer Research.
-a
879
M. Watanabe,
@
Y. Takabe,
T. Katsumata,
and T. Terasima
thymidine-3H at various times. After the pulse treatment
of
cells, cells were counted at intervals under a micro
In L-cells, the prolongation
of DNA-synthetic
phase
could be correlated with the remarkable action of bleomy
scope. The change of percentagelabeled cells in the G1- cm on the DNA synthesis and DNA molecules of cells.
treated population was almost like that of the untreated
Suzuki et al. (11) found that the DNA synthesis of HeLa
control through 17 hr after mitosis, except for slightly
cells was moderately inhibited by the antibiotic, while pro
tein synthesis was less affected. This finding was confirmed
higher values found at 13 and 15 hr. This result indicates
that bleomycin did not significantly affect the progression again by our study with mouse L-cells (21) in which bleo
of G1-treated cells through S phase, although the exit of mycin inhibited DNA synthesis most strongly and inhibited
cells from S phase was slightly delayed (less than I hr). protein and RNA syntheses, in that order, to a lesser ex
However, cell division of G1-treated cells was inhibited up tent. Bleomycin also affected cellular and isolated DNA
to 68 hr after inoculation of mitotic cells, and the 2nd wave molecules, producing single- and double-strand scissions in
DNA (17), decreasing the melting temperature (8), or lib
of labeled fraction was not observed. The results indicate
erating thymine base from DNA (6). In view of these ac
that cells exposed to bleomycin in G1 phase progressed
through S into G2 but were blocked at G2 phase with a tions of bleomycin on DNA, the results presented in Chart 2
can be explained as follows. The beginning of DNA-syn
considerable delay.
thetic phase was not affected by drug-caused strand scission
The G2 block was also observed when the asynchronous
of, or any other possible damage in, DNA molecules. How
population of L-cells was treated with bleomycin and Col
cemid (Chart 4). The accumulation
of mitotic cells was ever, the processes of genome replication would be delayed
shown in terms of the collection function (10). The value either by the degradation of DNA molecules and subse
obtained for untreated control increased linearly with time quent repair in cells (17) or by the possible inhibition of
after a short lag period which may correspond to the time enzymes related to DNA synthesis (7). Thus, the comple
phase in drug-treated cells was
needed for Colcemid to reach its active site. The value for tion of the DNA-synthetic
bleomycin-treated
cells closely followed the control curve delayed.
Another site of bleomycin action on the cell cycle tray
during the 1st 90 mm of exposure, then practically leveled
erse of L-cells is at the G2 phase. Other investigators (1, 18)
off, suggesting that cells were arrested in G2 phase (about
have reported that bleomycin interfered with cell prognes
90 mm before mitosis) and failed to enter mitosis.
sion during G2 phase. In our observation, more than 90
mm before mitosis, the cells failed to reach mitosis in the
DISCUSSION
presence of bleomycin (Chant 4). This finding would mdi
cate that the early pant of G2 phase is intrinsically sensi
In the presence of bleomycin, G1 cells entered the DNA
tive to bleomycin. In L-cells, the G2 block may be related to
synthetic phase at the same rate as the untreated control
the inhibition of protein or RNA synthesis required for cell
cells, but the progression of cells from S to G2 phase was division, since gross reduction of these macromolecular
delayed considerably (Chart 2). In contrast to our results,
syntheses was shown (21).
it was reported that bleomycin did not affect the DNA
It is particularly noted that the great prolongation in G2
synthetic phase of Chinese hamster cells (1, 18). These
phase was also observed when cells were exposed pulse
results may be specific for the Chinese hamster cell line, wise to bleomycin at G1 phase, as shown in Chart 3. This
since macnomolecular
syntheses were less inhibited in result is in agreement with our cinematographic
observa
Chinese hamster cells (18) than in other mammalian cells tions on bleomycin-treated
cells (4), in which synchronous
(11, 21).
culture of L-cells pulse treated with bleomycin at the G1-S
transition phase showed a remarkable cell division delay.
Incidentally, similar division delay has been observed in
cells treated with a DNA-attacking
agent during the G1
phase of the cell cycle. Ohara and Terasima (9) reported
that damages produced by mitomycin in the G1 phase of
HeLa cells brought about the marked prolongation of the
following S and G2 phases. This was assumed to be due to
irreversible damage to DNA of i cells. In this study, how
ever, it is less likely that damage to DNA is primarily re
Ui
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z
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sponsiblefor the observedG2 prolongation, sincethe DNA
synthetic phasewas minimally affected in G 1-treatedcells.
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-a
HOURS AFTER BLEOMYCIN ADDITION
Chart 4. Effect of bleomycin on progression of asynchronous cells into
mitosis. Bleomycin (100 @g/ml)and Colcemid (0. 1 .tg/ml) were added
to asynchronous cultures of I-cells at zero time. Then, samples were taken
at
intervals
and
prepared
for
determination
of mitotic
frequency.
Ordi
nate, accumulation of mitotic cells after addition of Colcemid in terms of
the collection function log ( 1 + mitotic index) (10).
880
Another possible explanation involves the damages in mi
totic apparatus. For example, damage to centriole, spin
dles, or nuclear membranes may have a relation to the
division delay in G1-treated cells. More extensive know!
edge of the mechanism of cell division, especially of the
division-essential
syntheses occurring
through the cell
cycle, might aid in elucidating the specific action of bleo
mycin on G2 phase of cells.
CANCER RESEARCH
VOL. 34
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Efftcts
ACKNOWLEDGMENTS
The authors thank Nippon Kayaku Co. Ltd., for the kind supply of
II.
bleomycin throughout this work. We are also grateful to Professor K.
Okuda, Chiba University School of Medicine, for his encouragement.
The skillful technical assistance of M. Yasukawa is acknowledged.
12.
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881
Effects of Bleomycin on Progression through the Cell Cycle of
Mouse L-Cells
Michinori Watanabe, Yosinobu Takabe, Takesi Katsumata, et al.
Cancer Res 1974;34:878-881.
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