[CANCER RESEARCH36, 2345-2349,July 1976]
The Significance of DNA Damage in the Cell Cycle Sensitivity
of Chinese Hamster Ovary Cells to Bleomycin
Judith M. Clarkson and Ronald M. Humphrey
University of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, Department of Physics, Houston, Texas 77030
SUMMARY
Chinese hamster ovary cells have been treated with bleomycin at various stages in the cell cycle. Mitotic cells, which
exhibit least survival, show the greatest amount of DNA
strand breakage and a marked inhibition of DNA replication
in the subsequent S phase. Strand-rejoining experiments
suggest that this is primarily due to differences in the
amount of damage produced in the DNA but does not exclude the possibility that cells at the various cell stages also
differ in their ability to repair this damage. DNA breakage
also occurs in cells incubated in bleomycin at 4 ~
INTRODUCTION
BLM, 2 a complex glycoprotein, is highly toxic to mammalian cells and is a potent cancer chemotherapeutic agent
(23, 24). A large amount of data is available concerning its
effect on cells in vitro and much of this has centered around
the differential sensitivity of various stages of the cell cycle.
Studies with CHO cells show that its killing efficiency is
greatest in M and then in G2 (1) and that, at concentrations
which cause little inhibition of leucine, uridine, or thymidine uptake cells accumulate in G2 (22).
However, one of the main targets for damage is DNA. TdR
uptake is inhibited to a much greater extent than uridine
and leucine uptake (9, 18) and DNA strand breakage by BLM
has been demonstrated both in vitro (4, 17, 21) and in vivo
(20). In vitro experiments have also shown that, under these
conditions, RNA is not degraded (5). This is consistent with
the findings of MOiler et al. (12), who showed that degeneration of nucleic acids and synthetic polynucleotides by BLM
depends on the liberation of thymine.
We have used strand breakage as a criteria for assessing
the amount of damage to DNA at different stages of the cell
cycle and have been able to correlate this with the reduction
in rate of DNA replication and subsequent survival.
MATERIALS
AND
METHODS
Cell C u l tur e and S y n c h r o n y T e c h n i q u e s . CHO cells were
used throughout and maintained as monolayer cultures in
1This work was supported in part by National Cancer Institute Grant CA04484 and Contract NO1 CM 61156.
= The abbreviations used are: BLM, Bleomycin; CHO, Chinese hamster
ovary; TdR, thymidine; HU, hydroxyurea;[~H]TdR,tritiated thymidine; BUclR,
5-bromodeoxyuridine.
Received August 4, 1975; accepted April 9, 1976
McCoy's 5A medium (Grand Island Biological Co., Grand
Island, N. Y.) with 20% fetal calf serum (Grand Island Biological Co.) as described by Humphrey et al. (8). Under
these conditions, the average cell generation time was
found to be 14 hr, of which the pre-DNA synthesis period
(G1) was 4 hr, DNA synthesis was 8 hr, and the post-DNA
synthesis period (G=), including mitosis, was 2 hr.
For cell cycle experiments, 107 cells were seeded into 32oz prescription bottles and 7.5 mM TdR was added to the
cultures for 9 hr, resulting in an accumulation of early Sphase cells. Following removal of the TdR block, the cells
progressed through the cell cycle, and 5 to 6 hr later a wave
of mitotic cells could be selected by the mitotic-shake
method (19). The resulting population has a greater than
85% mitotic index and was incubated in 2 mM HU for 9 to 12
hr, which allowed progression to the GI-S boundary (11).
After the cultures were washed and fresh medium was
added, the cells progressed into S phase. This synchrony
procedure and the times selected for treatments with BLM
are demonstrated in Chart 1. Meyn et al. (11) showed that,
subsequent to a TdR block, cells replicated only 75% of
their DNA, thus showing that this method of synchrony
results in the accumulation of cells that have partially completed DNA synthesis. At 0.5 to 1 hr post-TdR release, cells
were assumed to be in early to mid-S phase. Other stages
were: G2 period, 1 hr prior to mitotic shake-off; M, 1st halfhr following shake-off and before plating; early G~, 1 to 1.5
hr from mitotic shake-off; late G~, 0.5 hr prior to HU release;
early S, 1 hr post-HU release.
Drug T r e a t m e n t . The BLM was manufactured for clinical
use and supplied by Bristol Laboratories, New York, N. Y. It
was dissolved in 0.9% NaCI solution immediately before
use, and a concentrated solution was added to the growth
medium.
D N A R e p l i c a t i o n . DNA was uniformly labeled prior to
synchrony by incubating cells in 0.1 /~Ci/ml ['4C]TdR (50
mCi/mmole; Schwarz/Mann, Orangeburg, N. Y.) for 18 hr at
37 ~ Replication was estimated as rate of uptake of BUdR
and consequent increase in buoyant density in CsCI gradients (11). Following mitotic selection and incubation in
HU medium, the cells were washed twice and resuspended
in medium containing 50 /.~g BUdR p e r ml and 0.1 /~g
fluorouracil deoxyuridine per ml. After appropriate incubation times (up to 10 hr), cells-were harvested, lysed with
Sarkosyl, and prepared for equilibrium centrifugation. CsCI
solution was added to the cell lysate to give a 4.7-ml sample
with a density of 1.74 g / c u c m . This was centrifuged in a
Beckman 50.1 rotor at 33,000 rpm for 45 hr at 20 ~. The
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J. M. Clarkson and R. M. H u m p h r e y
Mitotic
fresh
TdR
H U Medium
8 - 10 Hours
Shake-off
medium
I
fresh
medium
I
Protocol
1
block
(9 Hrs)
I
I
I
I
uS
I
I
I
i i i
~,
I I
- '-!
~'I,I G2,,I
I
V
early-mid
s
I , I
n
',',
Ii J
-,,--',
---', , i',
I
I
n
L
n
',
G1
I ',
M
hours.
I
Ii,
Approximate
',,
cell stage.
s
a
V VV
G2
Time scale
marked in
?
VV
early
G1
late
G1
Treatment
times.
early
S
Chart 1. The procedure used for synchronizing cells. Also shown are the times chosen for treatment with BLM for the experiments in Chart 2 and Table 1.
percentage of DNA replicated was d e t e r m i n e d f r o m the
p r o p o r t i o n of ~4C radioactivity b a n d i n g in the hybrid density
region of the gradient.
P r o g r e s s i o n of C e l l s into S P h a s e . Cells were synchronized by the p r o c e d u r e just described, 104 mitotic cells were
plated in 6-cm Petri dishes, and HU m e d i u m was added as
before for 8 to 10 hr. To 1 set of plates, [:~H]TdR (specific
activity, 2 C i / m m o l e ) was added to give a c o n c e n t r a t i o n of
0 . 5 / ~ C i / m l for the last hr of i n c u b a t i o n . The r e m a i n i n g samples were washed free of HU and [3H]TdR m e d i u m was
added for 1 , 2 , 4, or 6 hr. The cells were washed wi t h 0.9%
NaCI solution, fixed in 95% alcohol, and subbed with 1%
bovine serum a l b u m i n . A u t o r a d i o g r a p h s were made with
Ilford liquid e m u l s i o n K5 and were exposed for 5 days. The
plates were then stained with Giemsa, and the percentage
of cells covered by grains was estimated.
S u c r o s e G r a d i e n t s . For e x p e r i m e n t s with a s y n c h r o n o u s
cultures, the cells were incubated o v e r n i g h t in 0.1 /~Ci
['4C]TdR per ml in 6-cm Petri dishes. Where s y n c h r o n o u s
p o p u l a t i o n s were required, sufficient cells were obtained by
shaking M-cells from an a s y n c h r o n o u s culture prelabeled
wit h 0.1 /~Ci/ml [~4C]TdR.
F o l l o w i n g drug t r e a t m e n t and s u b s e q u e n t i n c u b a t i o n periods, the cells were trypsinized in 0.1% trypsin for 5 min at
37 ~ washed in S o l u t i o n A (26), and resuspended in 0.15 M
NaCI and 0.015 M s o d i u m citrate to give a final concentration of 1 to 2 x 10 .~cells in 0.5 ml. This mixture was layered
o n t o a 35-ml alkaline sucrose gradient (10) to w h i c h 0.5 ml
of lysis solution (0.5 M NaOH-0.02 M EDTA-0.1% Triton X100) had been added. Lysis was for 6 hr at room temperature and c e n t r i f u g a t i o n was at 12 K rpm for 9.5 hr at 20 ~.
Fractions were collected from the top of the gradient and
precipitated with 10% t r i c h l o r o a c e t i c acid.
The gradients were calibrated with DNA f r o m T4 (72 S)
and SV40 virus (46 S).
RESULTS
Chart 2 and Table 1 represent 2 e x p e r i m e n t s s h o w i n g the
effect on DNA replication of treating cells with BLM. Following release f r o m the HU block, the cells move into S phase
and their rate of DNA replication has been m o n i t o r e d by
uptake of BUdR, resulting in an increase in the buoyant
2346
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MHOSlS
Eor/y S
-6
75
o
o
~
o
. ,o
_
50
'-'
25
o
o
oBleomyr (100~g/ml)
2
4
6
8
y oIll
10
2
,oo Bl
Bleioomycm(25~g/ml
myc,,n(lOO#g/ml),
4
6
8
10
Hours p0st-HU Rele0se
C h a r t 2. T h e effect on D N A r e p l i c a t i o n after t r e a t m e n t w i t h B L M in e a r l y S
or m i t o s i s . Cells w e r e p r e l a b e l e d w i t h [I~C]TdR a n d w e r e s y n c h r o n i z e d by
e x c e s s T d R , m i t o t i c s e l e c t i o n , and i n c u b a t i o n in HU m e d i u m . T h e cells w e r e
t h e n t r a n s f e r r e d to B U d R m e d i u m for v a r i o u s i n c u b a t i o n times. B L M was
a d d e d for 30 min e i t h e r to m i t o t i c cells b e f o r e p l a t i n g in HU or to the B U d R
m e d i u m 1 hr after its a d d i t i o n to the cells (early S cells).
Table 1
DNA replication following treatment of cells with 100 i~g/ml BLM
for 30 min at different stages of the cell cycle
Cells prelabeled with [~4C]TdR were synchronized in excess TdR,
mitotically selected, incubated in HU medium, and transferred to
BUdR medium for 4 or 8 hr. BLM was added for 30 min either
following release from TdR (S), 1 hr prior to the mitotic shake (G2),
immediately following mitotic selection (M), 1 hr following plating
(early G1), or 0.5 hr prior to release from HU (late GI); see Chart 1.
Untreated controls were synchronized by the same procedure.
DNA replication (% of total)
Position in the cell cycle at
time of treatment
4 hr post-HU
release
8 hr post-HU
release
S
G2
M
Early G,
Late G~
Control
49
46
14
50
46
54
90
84
30
91
87
92
density of DNA in CsCI gradients. It is evident from this data,
as in earlier studies (11), that DNA replication proceeds at a
c o n s t a n t rate and reaches 95% c o m p l e t i o n 8 hr after removal of the HU. In Table 1 , 2 time intervals post-HU release
CANCER RESEARCH VOL. 36
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Research.
DNA Damage in CHO Cells Exposed to Bleomycin
have been chosen f o l l o w i n g treatment of cells at different
stages of the cell cycle with 100 /xg of BLM per ml.
The a m o u n t of DNA replicated in 4 or 8 hr is very similar
for control cells and those incubated with BLM for 30 min in
early G1, late G~, S, or G~. The only stage that is sensitive at
this c o n c e n t r a t i o n and subsequently results in a reduced
rate of DNA replication is mitosis. This is also illustrated in
Chart 2, where it can be seen that 2 5 / x g / m l at this stage is
also effective in reducing the rate of DNA replication. In
Table 2, we have c o n s i d e r e d the possibility that this effect is
a result of cells being blocked n G~ and not entering S
phase. In the control samples, 94% of the cells become
labeled in the 1st hr post-HU release. Cells treated with BLM
during mitosis are s o m e w h a t slower entering S, but by 2 hr
more than 70% of the cells are labeled f o l l o w i n g either 25 or
100 /.~g of BLM per ml and, at 6 hr, more than 85% are
labeled. This delay is not sufficient to a c c o u n t for the considerably reduced a m o u n t s of DNA replicated. In addition,
we have shown that no DNA synthesis has taken place in the
presence of HU in either the treated or c o n t r o l samples.
Charts 3 to 5 show the a m o u n t of breakage in DNA molecules at different stages of the cell cycle as d e m o n s t r a t e d by
molecular-weight studies on alkaline sucrose gradients. In
Chart 3, the effect of 2 5 / x g / m l for 30 min is c o m p a r e d in G~,
S, and mitotic cells. It is evident that the size of the DNA in
the untreated c o n t r o l s is very similar in all stages of the cell
Table 2
The progression of cells into S phase following BLM treatment in
mitosis and incubation in HU medium for 10 hr
[:~H]TdR was added for various lengths of time and autoradiographs were made. The results are expressed as percentage of
cells labeled.
Time in ['~H]TdR medium
Control
25/~g/ml
100/xg/ml
0
94
93
96
99
0
66
71
82
87
0
65
72
76
85
1 hr prior to HU release
0-1 hr post-HU release
0-2 hr post-HU release
0-4 hr post-HU release
0-6 hr post-HU release
Sed~menlohon
--- -
Contro/
Bleomycin (Z5tzg/ml
--
Bleomycm
, 30 min }
3 0 r a i n , f r e s h reed#urn
2hr
Mitosis
GI
#
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10
20
30
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10
20
30
Fraction Number
Chart 4. DNA strand rejoining of mitotic and G~ cells in the presence of
Colcemid following BLM treatment. Mitotic cells were collected from an
asynchronous culture labeled with 0.1 /~Ci ['4C]TdR per ml. G, samples were
incubated 2 hr further before treatment. BLM, at 25 /zg/ml, was added to
experimental plates and Colcemid, at 0.06/~g/ml, was added to all samples.
After 30 min, the BLM was washed off and fresh medium plus Colcemid was
added to 1 of each sample for 2 hr more. Mitotic selection was arranged so
that all samples could be harvested at the same time, lysis was for 6 hr, and
samples were spun 9.5 hr at 12,000 rpm. One fraction covers 9.3 S.
Sed/mentohon
37 ~
4~
-----
25 tzg/ml
30ram
.~
'~
i
--.- -
25tzg/ml
25/xg/ml
....
Control
30rain
3hr
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~s
o
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ac:
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-\
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.,
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./
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I0
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',.
/
30
I
I0
20
30
Fraction Number
S
Sed/menfghon
m
o
I0
.....
Conlrol
--
Bteomycm
Milosi5
(25pg/ml,O 5 hr)
Chart 5. Dependence on 37~ temperature for DNA strand breakage by
BLM. Cells were prelabeled with 0.1 /~Ci ['4C]TdR per ml and mitotic cells
were collected at various times for treatment with 25/~g BLM per ml at 4 or
37~as shown. Lysis was for 6 hr and centrifugation was at 12,000 rpm for 9.5
hr. One fraction covers 9.3 S.
S
~
5
o
cr
0
10
20
IO
2O
Fraction Number
Chart 3. Cell stage sensitivity to DNA strand breakage by BLM. An asynchronous cell culture was labeled with 0.1 /~Ci ['4C]TdR per ml and shaken at
various times to give mitotic cells. Two samples were incubated in HU
medium for 6 hr and then allowed to proceed into S phase for 2 hr. Two
samples were incubated for 8 hr in HU and treated with BLM in the same
medium (G~ sample). At this time, 2 further samples were collected and to 1
of each sample BLM was added to give 25 /~g/ml for 30 rain. The BLM was
then washed off and the cells were immediately harvested and lysed on top of
the gradients for 6 hr. Centrifugation was for 9.5 hr at 12,000 rpm, 1 fraction
covering 9.3 S.
I0
20
cycle, as d e m o n s t r a t e d by Lett et al. (10) and H u m p h r e y et
al. (7). In each instance, it peaks in Fraction 18 and 19,
agreeing well with reported values of 165 S for c o n t r o l DNA
(10). For G1 and S phase cells, B L M t r e a t m e n t results in
breakage of the DNA from this c o n t r o l size to 100 and 105 S
in the peak fraction. However, in m i t o t i c cells a larger red u c t i o n in m o l e c u l a r w e i g h t is evident; the peak being 80 S.
This cell cycle effect was also observed for 1 0 0 / ~ g / m l where
more total d a m a g e o c c u r r e d . T r e a t m e n t in G1 resulted in
breakage to 55 S and in M to 35 S.
DNA strand rejoining after removal of BLM from the cell
c u l t u r e is s h o w n in Chart 4 for GI and M-cells. Mitotic cells
were treated either i m m e d i a t e l y on c o l l e c t i o n or after a 2-hr
i n c u b a t i o n period (G~ cells). B L M and C o l c e m i d were added
and, 30 min later, this medium was replaced by fresh medium plus Colcemid (to prevent M-cells from moving into
JULY 1976
2347
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J. M. Clarkson and R. M. H u m p h r e y
G1). These data show that, despite the large discrepancy in
the initial amounts of damage, most of the DNA strand
breaks are rejoined within 2 hr for both cell stages.
In Chart 5, the effect of treating cells at 4 ~ is shown. At this
lowered temperature, the amount of strand breakage increases with incubation time and after 3 hr is comparable to
that after 30 min at 37 ~. However, after 3 hr at 37 ~, it is
evident that many of the breaks are rejoined (Chart 4) even
in the presence of BLM.
DISCUSSION
BLM has been shown to have a cell stage-specific effect
on the survival of CHO cells (1). In this report, data are
presented with the intention of elucidating this cell cycle
effect, particularly with reference to the significance of
damage to DNA. We have attempted to determine (a)
whether this is 1 of the primary targets for damage; and (b) if
this is so, whether cell cycle-dependent survival is correlated With differences in the amount of DNA damage induced or with differences in the DNA break-rejoining capacity of cells at different stages.
Inhibition of DNA synthesis by BLM has been demonstrated by Kunimoto et al. (9) and Suzuki et al. (18), and we
have confirmed this. By choosing the appropriate concentration (100/~g/ml) of BLM, we have, in addition, been able
to show a differential effect on subsequent DNA replication,
depending on the cell phase treated. At this concentration,
very little effect on S, G2, or G1 cells is evident, but the rate
of DNA replication following treatment of mitotic cells is
markedly inhibited (Chart 2; Table 1) and appears to terminate at 70% completion following 25 # g / m l and at 60%
completion following 100 /~g/ml. This agrees well with the
survival data of Barranco and Humphrey (1), where a 2-fold
decrease in survival of M-cells over the next most sensitive
stage, G2, was found. Nagatsu et al. (14) and Watanabe et al.
(25) showed that the greatest progression delay occurred in
G2 and, for continuous treatments with BLM, Tobey (22)
found that entry into S phase was affected less than entry
into mitosis with the result that cells tended to accumulate
in G=. Recently, Hittelman and Rao (6), using the technique
of premature chromosome condensation, showed that only
35% of the chromosomal gaps produced by 25 #g of BLM
per ml applied in G= could be repaired and that 44% of the
cells were arrested in G2. Our data for the effect on subsequent DNA replication do not show G= to be any more
sensitive than S or G1. However, since many of the cells are
arrested at this stage and do not reach mitosis, an accurate
estimate of the relative sensitivity of cells treated in stages
prior to mitosis is not possible.
DNA breakage by BLM has been demonstrated both in
vitro (4, 17, 21) and in vivo (20), and we have used this as a
criterion for the amount of damage induced in DNA. In
Chart 3, the results are shown following treatment of cells at
different stages in the cell cycle. Compared with G~ and S, it
is clear that mitosis is the most sensitive stage with regard
to DNA damage, and this finding correlates well with the
DNA replication data presented here and with the cell survival data of Barranco and Humphrey (1). After 30 min of
incubation with the drug, it is possible that the cell has
2348
already repaired some of the damage produced and, thus,
we are looking at the net result of 2 processes, induction of
breaks in DNA and their rejoining. DNA strand rejoining
after removal of BLM from a cell culture has already been
demonstrated (16, 20) and the results in Chart 4 confirm
this. A 30-min treatment with 25 /~g BLM per ml was followed by a 2-hr recovery period in fresh medium (all in the
presence of Colcemid). In the case of G~ cells, DNA strand
rejoining is completed in this time. The mitotic cells still
have a small percentage of their DNA in the form of small
strands, although most of the damage has been repaired. It
was shown by Humphrey (7) that strand rejoining following
X-irradiation was considerably slower in M-cells than in Sphase cells, although the M-cells eventually recovered to
the same extent. It is possible that the slight difference
between the profiles of the G~ and M-cells 2 hr after treatment is the result of a similar difference in rate of recovery.
However, it is evident from these data that the most significant difference in the response of these 2 cell stages is
manifested in the initial levels of damage and not in the
capacity for rejoining of DNA strand breaks. Data from
Escherichia coli indicate that sensitivity to BLM is linked to
cell membrane permeability (2), and Nakashima et al. (15)
found an enhanced effect of BLM in the presence of pentamycin, which they suggest increases permeability of the cell
to the drug. It is therefore possible that breakdown of the
nuclear membrane in preparation for mitosis is significant
in this respect. Autoradiographic studies by ":-,.Jiimoto (3)
show that 14C-labeled BLM becomes concer,~rated o r the
nuclear membrane of mouse tumor cells. The resolts shown
in Chart 5 make it unlikely that BLM is actively transported
into the cell and that a difference in cellular metabolism
throughout the cell cycle would be significant in this respect. This contrasts with our data (J. M. Clarkson and R.
M. Humphrey, unpublished data) for adriamycin, where
strand breakage occurs only at 37 ~.
It is interesting that 25 ~g and, especially, 100 ~g of BLM
per ml do cause strand breakage in DNA at all stages of the
cell cycle but that a certain level of damage can be tolerated
and/or repaired so that, even when the drug is applied
during the same S phase, the rate of DNA replication is not
reduced (Chart 2). Similar results were obtained by Myer
and Strauss (13)for methyl methanesulfonate-treated HEp-2
cells. Single strand breaks were produced in the DNA of
these cells which were then able to proceed through 1
subsequent S phase but were unable to complete a 2nd
round of DNA replication.
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Survival and Cell Progression in Chinese Hamster Cells in Vitro. Cancer
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1974.
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D N A D a m a g e in C H O C e l l s E x p o s e d to B l e o m y c i n
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1976 American Association for Cancer
Research.
The Significance of DNA Damage in the Cell Cycle Sensitivity
of Chinese Hamster Ovary Cells to Bleomycin
Judith M. Clarkson and Ronald M. Humphrey
Cancer Res 1976;36:2345-2349.
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