[CANCER RESEARCH 42, 4744-4752,
0008-5472/82/0042-OOOOS02.00
November 1982]
Relationship between Aberrant DNA Replication and Loss of Cell
Viability in Chinese Hamster Ovary CHO-K1 Cells
David M. Woodcock,1 Julian K. Adams, and Ian A. Cooper
Haemato/ogy Research Unit. Cancer Institute, 481 Little Lonsdale Street, Melbourne.
3000. Victoria, Australia
aberrant form of DNA synthesis has to cell killing by these
agents, we have used the CHO-K1 cell line and have, within
We have previously presented evidence that a transient block
the same experiment, simultaneously examined the effects of
to DMA replication induces an aberrant form of DNA synthesis.
2-hr pulses of inhibitors of DNA replication (both direct and
The most feasible explanation for this data is that the block to indirect inhibitors) on loss of cell viability assayed by loss of
DNA replication results in some segments of the chromosomal
colony formation ability as well as the relative extent of this
DNA being replicated more than once in a single cell cycle.
aberrant form of DNA replication.
This form of aberrant DNA synthesis was demonstrated to
The method used to estimate the relative extent of this
occur following direct inhibition of DNA replication by 1-ß-o- aberrant DNA replication is a modification and extension of the
arabinofuranosylcytosine
or 9-/S-D-arabinofuranosyladenine
or method used previously (32, 34, 35). The logical basis of this
after indirect inhibition with cycloheximide. We have proposed
method has been described in detail (32, 34, 35). Briefly,
mechanisms whereby this phenomenon could induce chromo
semiconservative replication of the chromosomal DNA will re
some damage and cell death. In this paper, we present data on
sult, at the end of each S phase, in all DNA duplexes present
the relationship between this aberrant form of DNA replication
in the cell containing one parental (template) strand and one
and the loss of cell viability. Using Chinese hamster ovary CHOdaughter strand which was synthesized during that cell cycle.
K1 cells growing as monolayer cultures, we have simultane
During a single cell cycle, normal semiconservative DNA rep
ously monitored the loss of cell viability as measured by colony
lication cannot produce any piece of DNA duplex, both strands
formation and the relative extent of this aberrant DNA replica
of which were synthesized during that one cell cycle. The
tion induced by 2-hr pulses of a series of concentrations of
method to demonstrate aberrant double replication of chro
inhibitors of DNA replication. We have found that, with either
mosomal DNA segments is, in essence, a method to demon
direct inhibition of DNA replication with 1-/?-D-arabinofuranostrate the production, following a transient block to DNA repli
sylcytosine or with indirect inhibition with cycloheximide,
cation, of DNA duplex segments, both strands of which had
pulses of inhibitor administered to Chinese hamster ovary cells
been synthesized in that one cell cycle.
at increasing concentrations induced a progressive increase in
In the experiments reported here, asynchronously growing
the extent of this aberrant DNA replication which paralleled the monolayer CHO2 cells were labeled with a 1-hr pulse of [3H]
increase in cell killing.
dThd, the cells washed to remove label, and the label chased
for 1 hr in fresh medium. At this point, an inhibitor of DNA
replication was added, and 2 hr later the cells were rinsed. The
INTRODUCTION
cells were then incubated for 4 hr in fresh medium containing
In any eukaryotic cell, each segment of the chromosomal
BrdUrd. The BrdUrd was present to density label the DNA
DNA must be replicated once and once only in each cell cycle
strands synthesized after the period of DNA replication inhibi
(28). Failure of one segment to replicate would result in the tion. If the transient block to DNA synthesis induced aberrant
sister chromatids being unable to separate at mitosis. Repli
reinitiation of DNA replication in DNA segments replicated
cation of segments more than once in a single cycle would
shortly before the period of DNA replication inhibition (which
under these conditions were labeled with [3H]dThd), DNA du
mean instability in the size of the genome, open replicative
forks [and hence probable failure of chromosome condensation
plexes would be formed which contained BrdUrd-containing
daughter strands and 3H-labeled template strands. With this
(21, 25)] when the cell reaches metaphase, and uncontrolled
segregation of chromosomes of mitosis (16, 21, 36). (The
experimental protocol, this aberrant reinitiation is demonstrated
cellular consequences of double replication of DNA segments
by purifying the hybrid density DNA duplexes (light-heavy
are argued more fully in Ref. 33.) Hence, any process that
duplexes with BrdUrd-containing
daughter strands) using 3
results in some segments of the chromosomal DNA being
cycles of neutral CsCI gradients and separating and banding
replicated more than once in a single cell cycle is likely to have
the normal density template and BrdUrd-containing daughter
major deleterious effects on a eukaryotic cell. We have previ
strands of the purified light-heavy duplex DNA on alkaline CsCIously presented evidence that a transient block to DNA repli
Cs2SC>4gradients. The greater the relative frequency of aber
cation induces just such an effect (32, 34, 35), and we have
rant reinitiation of DNA replication compared to normal initiation
argued that this might be the cause of chromosome aberrations
2 The abbreviations used are: CHO, Chinese hamster ovary; dThd, 2'-deoxyinduced by inhibition of DNA replication and of the consequent
ribosylthymine; BrdUrd, 5-bromo-2'-deoxyribosyluracil;
PBS-EDTA, phosphateloss in cell viability (33).
buffered saline with EDTA (0.15 M NaCI-0.7 mw KCI-4.3 mM K2HPCv0.02%
In order to further elucidate what relevance, if any, this
glucose-0.02% Na2EDTA); BSS, balanced salt solution (0.8% NaCI-0.04% KCIABSTRACT
' Recipient of a grant from the National Health and Medical Research Council
of Australia. To whom requests for reprints should be addressed.
Received January 18, 1982; accepted August 5, 1982.
4744
0.1% glucose-0.02% phenol red-0.28% NaHCO3-0.015 M 4-(2-hydroxethylH
piperazineethanesulfonic
acid buffer-gentamicin (20 units/ml); FdUrd, 5-fluoro2'-deoxyribosyluracil;
dCyd, 2'-deoxyribosylcytosine;
ara-C. 1-/3-o-arabinofuranosylcytosine; ara-G, g-yS-o-arabinofuranosylguanine.
CANCER
RESEARCH
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VOL.
42
Aberrant DNA Replication and Loss of Cell Viability
of DNA replication,
the higher the proportion
of BrdUrd-con-
taining daughter strands which will be synthesized off
[3H]-labeled template strands and hence the higher the specific
activity (3H cpm/fig DNA) of the template strands (light strands
in alkaline density gradients of the purified light-heavy DNA).
Hence, this methodology allows an estimation of the relative
extent of aberrant reinitiation. However, this methodology does
not provide any quantitative estimation of the extent of aberrant
DNA replication since it is not possible with this method to
estimate the specific activity of those DNA strands synthesized
during the [3H]dThd labeling period.
Note that this method to demonstrate aberrant reinitiation
should not be confused with another aberrant form of DNA
which has been reported previously (19, 27). DNA isolated
from mammalian cells growing in the presence of BrdUrd for
less than a single cell cycle contains under certain circum
stances a small fraction of doubly BrdUrd-substituted
DNA.
This DNA is an extraction artifact arising from branch migration
in the DNA of replicating replicons (19, 27). Since both daugh
ter strands at any given replication fork are synthesized at the
same time, both daughter strands will have the same compo
sition and will thus be of either light or heavy density, depending
on whether BrdUrd was present at the time or not. Hence,
branch migration during extraction in the DNA of replication
forks could produce either light-light or heavy-heavy artifactual
duplex segments. Under no labeling protocol could this type of
extraction artifact produce light-heavy duplex segments.
Therefore, this phenomenon cannot interfere with the experi
ments described in this paper which involve the isolation of the
light-heavy DNA fraction.
MATERIALS
radioactive medium was removed carefully using vacuum aspiration.
The cells were then rinsed with BSS plus 2% fetal calf serum, which
was similarly removed. The [3H]dThd was then chased for 1 hr in
normal growth medium supplemented with Colcemid (0.2 jug/ml) to
prevent any cells entering a second cell cycle during the time of the
experiment. Following the 1-hr chase, inhibitor was added to test
cultures. After 2 hr in the presence of inhibitor, the inhibitor-containing
medium was carefully removed by aspiration, the cells were washed
with BSS plus 2% serum, and growth medium containing 10¡IMBrdUrd,
1 /¿M
FdUrd, 10 JUMdCyd, and Colcemid (0.2 ng/m\i was added. The
BrdUrd-containing
medium was added to control cultures at the time
inhibitors were added to the test cultures, this being done at this time
because in test cultures there was a very low rate of DNA replication
during the time the inhibitors were present. After 4 hr in the BrdUrdcontaining medium, cells were collected by rinsing the flasks with PBS
and using pronase in PBS-EDTA to disaggregate the monolayers. The
concentration
of Colcemid used (0.2 /¿g/ml) was found to be the
optimal level for these cells under these conditions for maximal fre
quencies of cells arrested in metaphase after a 6-hr treatment (not
illustrated).
Purification of Hybrid Density DNA Fraction and Alkaline Gradient
Analysis. DNA extraction and purification was performed as described
previously (32, 35). After shearing of DNA to approximately 6000 base
pairs (32, 35), the light-heavy DNA fraction was purified using 3 cycles
of neutral CsCI gradients as described previously except that in earlier
experiments 2 cycles of neutral gradients were used. Also, 20 fil of 3%
Sarkosyl (Ciba-Geigy Corp.) was included in each gradient. The strands
of the purified light-heavy DNA were separated and banded in alkaline
CsCI-Cs2SO4 gradients which permitted both the normal density and
the heavy BrdUrd-substituted
strands to be well separated yet for both
to band well within the gradient. This procedure was as described by
Kowalski and Cheevers (12) except that the amounts of all components
were 1.25 times those used by these authors. For the estimation of the
specific activities (3H cpm/^g DNA) of the light strands from the purified
light-heavy
AND METHODS
Chemicals and Radiochemicals. All nucleosides, nucleoside ana
logs, cycloheximide, and Colcemid were purchased from the Sigma
Chemical Co., St. Louis, Mo. Radiochemicals were from The Radiochemical Centre, Amersham, United Kingdom. All other chemicals
were analytical reagent grade.
Cell Line and Culture Conditions. Experiments used cells of the
CHO-K1 cell line growing as monolayers in the a modification of
Eagle's minimum essential medium (Flow Laboratories, Rockville, Md.)
supplemented with 10% fetal calf serum (Commwealth Serum Labo
ratories, Parkville, Victoria, Australia). Cell cultures were routinely
monitored for Mycoplasma contamination (29).
Cell Viability Assays. Following specific drug treatments of CHO
cells, the flasks were rinsed with PBS-EDTA and monolayers of cells
were disaggregated with pronase (0.11 /ng/ml) (Calbiochem-Behring
Corp., La Jolla, Calif.) in PBS-EDTA. Following a wash with BSS and
DNA fraction,
the amount of DNA pooled from the light-
heavy fraction of the third cycle of neutral CsCI gradients was estimated
from the A260 values of the pooled fractions. The amount of DNA in
each alkaline CsCI-Cs2SO4 gradient was equalized to 50 jug/gradient
using nonradioactive BrdUrd-substituted
DNA. The assumption was
made that there were equal recoveries of the [3H]DNA from the alkaline
gradients in each experimental set. Because of the similarity of the A26o
profiles in each set of alkaline gradients ([3H]DNA plus unlabeled
carrier), it was considered that this assumption was justifiable. For the
calculation of specific activities, recoveries of DNA were all assumed
for simplicity to be 100%. The BrdUrd-substituted
carrier DNA was
prepared by growing CHO cells overnight in normal growth medium
supplemented with 10ftw BrdUrd, 1 ¡J.M
FdUrd, and 10 ^M dCyd.
Effectiveness of the Chase Procedure for [3H]dThd Pulse Label
ing. To check the effectiveness for the [3H]dThd pulse labeling used in
the above methodology to assess the extent of aberrant DNA replica
tion, [3H]dThd was added to cultures of CHO cells and, 1 hr later, cells
2% fetal calf serum, cells were resuspended in BSS plus 2% serum
and counted using hemocytometer counting chambers. After 3 steps
of dilution of 1 in 10 in BSS plus 2% serum, an average of 833 cells in
BSS plus 2% serum (<2 ml) were diluted to 25 ml with culture medium.
Aliquots (3 ml) of this cell suspension (average of 100 cells) were
transferred to 5-cm plates and incubated at 37° for 7 days in a
were rinsed and incubated in fresh medium as in the above procedure.
At one-half-hr intervals from 0 to 1.5 hr thereafter, BrdUrd (10 ¿IM),
humidified atmosphere of 5% O2, 10% CO2 and 85% N2. This resulted
in 7 plates/point.
To stain the colonies, the plates were rinsed with
0.9% NaCI, fixed with buffered formaldehyde solution (0.4% NaH2PCv
H2O-0.65% Na2HPO„-4%HCHO) and stained with 0.01 % crystal violet.
cellularly or still free in the medium due to any deficiency
Colonies with more than 50 cells were scored
efficiencies of control cultures were typically 75 to
Labeling Procedures to Estimate Aberrant
[methyl-3H]dThd (22 Ci/mmol) was added at 1
as viable. Plating
85%.
DNA Replication.
juCi/ml to growth
medium of asynchronous cultures of CHO cells which were usually 60
to 80% confluent at the time of the experiment. One hr later, the
NOVEMBER
1982
FdUrd (1 JUM)and dCyd (10 JIM) were added to portions of the culture.
Cells were incubated in culture medium with the BrdUrd for 3 hr before
being collected. DNA was extracted and banded in alkaline CsCI
gradients (35). All or most of [3H]dThd remaining unincorporated intrain the cell-
rinsing procedure and hence available for incorporation into DNA at
the beginning of the BrdUrd density-labeling period would be incor
porated into BrdUrd-containing strands during the 3 hr with the BrdUrd.
Therefore, the proportion of total 3H label at heavy-strand density in
the alkaline CsCI gradients is a measure of the effectiveness of the
chase procedure. The results of such an experiment are illustrated in
Table 1. With these cells, the proportion of 3H label at heavy-strand
density remained effectively the same after only 0.5 hr of chase. Hence,
a 1-hr chase in fresh medium provides an effective chase for the
4745
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D. M. Woodcock et al.
[3H]dThd labeling procedure
used in the methodology
to estimate the
extent of aberrant DNA replication. Note, however, that, even if the
chase procedure is not totally effective, this will not invalidate the
estimation of aberrant replication since unincorporated [3H]dThd pres
ent in cells after drug treatment will be incorporated into the Brdllrdcontaining daughter strands and hence cannot affect the specific
activity of the light-density template strands.
RESULTS
In the experiments reported in this paper in which effects of
DNA synthesis inhibition on both DNA replication and cell
viability were determined simultaneously, many more cells were
required for the former section of each experiments than for
the latter. To ensure that the cells used in both parts of the
experiment were growing in exactly the same way, cells were
subcultured into the different-sized flasks at the same time
from the same cell stock at equivalent cell densities with the
same batch of medium which was dispensed in volumes proTable1
Effectivenessof chaseprocedurefor[*H]dThdlabeling
CHO cells were incubated with [3H]dThd for 1 hr and rinsed, and then fresh
medium was added as in the procedure to estimate the extent of aberrant DNA
replication. BrdUrd plus FdUrd and dCyd were added to the medium at one-halfhr intervals between 0 and 1.5 hr after the end of the 3H-labeling period. After 3
hr in the presence of BrdUrd, cells were collected, and their purified DNA banded
in alkaline CsCI gradients.
% of total 3H cpm in heavy strands
Time of chase (hr)
0
0.5
1
1.5
1.31
0.61
0.65
0.47
portional to the growth area of the flasks. The cell plating to
assess viability was done at the same time as the other part of
the experiment in which the extent of the aberrant DNA repli
cation was examined. Sufficient cells were inoculated into each
flask for the cells to be 60 to 80% confluent at the time of the
experiment.
An experiment to examine the extent of aberrant DNA repli
cation induced by 2 hr pulses of a series of increasing concen
tration of ara-C is illustrated in Chart 1. Asynchronously grow
ing, log-phase CHO cells were labeled with a 1-hr pulse of
[3H]dThd. After rinsing the cells and chasing the label for 1 hr
in fresh medium, the ara-C was added to test cultures. Follow
ing 2 hr in the presence of ara-C, these cell cultures were
washed, and medium containing BrdUrd was added so that the
DNA strands synthesized after the ara-C pulse would be density
labeled. Density labeling of control cultures was commenced
at the time ara-C was added to the test cultures. This was done
at this time because, during the 2 hr ara-C was present in test
cultures, there was effectively no DNA synthesis. ara-C at the
concentrations used inhibited [3H]dThd incorporation into coldacid-insoluble material by 95% or more (not illustrated). Cells
were collected after 4 hr in BrdUrd medium. Colcemid was
present in all cultures from the time of the end of [3H]dTHdlabeling procedure onwards in order to prevent cells from
entering a second cell cycle. (See also Charts 3 and 4 and
discussion thereof for confirmation of this point.)
At the same time, ara-C was added to equivalent cultures of
CHO cells for assessment of the effects of the drug on cell
viability. After 2 hr with the drug, cells were rinsed, the cell
monolayers were disaggregated with pronase, and the single
B
D
1-5
Chart 1. Estimating the relative extent of
aberrant DNA replication after a 2-hr pulse of
ara-C. Asynchronously
growing CHO cells
were labeled with a 1-hr pulse of [3H]dThd and
after a 1-hr chase in fresh medium. ara-C was
added to portions of the culture at 10~5 M
(Column ß),1CT4 M (Column C), and 10~3 M
(Column D). After 2 hr with ara-C, cells were
rinsed and incubated in fresh medium contain
ing BrdUrd, FdUrd, and dCyd. Control cells
(Column A) were transferred to the BrdUrdcontaining medium after the 1-hr chase. The
light-heavy DNA fractions from control and
ara-C pulsed cells were purified using 3 cycles
of neutral CsCI gradients. Rows 1 to 3, 3
successive steps in the purification procedure.
The strands of the purified light-heavy DNA
fractions were separated and banded in alka
line CsCI-CszSO» gradients (Row 4). Bars.
fractions pooled from each cycle of neutral
CsCI gradients. Left and right arrows in each
of the neutral CsCI gradients, respectively, the
CsCI densities of 1.75 and 1.70 g/ml. The
bottoms of all gradients are to the left. The
fractions on the light density side of those
pooled from the third cycle of neutral CsCI
gradients (flow 3) were filtered and counted.
In all cases, any remaining contamination with
light-light DNA of the light-heavy DNA would
have contributed an insignificant number of
counts to the alkaline density gradients.
1005-
0
04-
02600
04-
200
0
400
I-200
•¿â€¢â€¢â€¢
*...•"
10
20
1
10
20
fraction
4746
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400 X
1
10
10
20
20
0
number
CANCER
RESEARCH
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VOL. 42
Aberrant DNA Replication and Loss of Cell Viability
cell suspension was diluted for plating. In control experiments
(not illustrated), it was found that 2-hr ara-C treatment had the
same effect on cell viability whether cells were subcultured for
viability plating immediately after the ara-C treatment or if the
cells were rinsed free of inhibitor and then incubated for 4 hr
in fresh medium before subculturing for plating. Thus, there
seem to be no equivalent to X-ray potentially lethal-damage
repair operating after ara-C treatment with these cells.
In assessing the relative extent of aberrant DNA replication,
DNA was purified from the cells collected after the 4 hr in
BrdUrd-containing medium, and the light-heavy DNA fraction
(the DNA synthesized during the 4-hr period following the araC pulse) was purified using 3 cycles of neutral CsCI gradients
(Chart 1, Rows 1 to 3). The purified light-heavy duplex DNA
was banded in alkaline CsCI-Cs2SO4 gradients in which the
DNA strands separated, the normal-density (template) strands
banding towards the top (right) of the gradients and the BrdUrdcontaining (daughter) strands banding towards the bottom (left)
of the gradients (Chart 1, Row 4). The relative specific activities
of the template strands from the light-heavy DNA from control
cells and cells treated with 2-hr pulses of ara-C at concentration
of 1CT5 to 10~3 M are illustrated in Chart 2A. (The specificity
activity of template strands from control cell DNA in this exper
iment was 56.4 cpm/jug light-strand DNA.) Chart 2A also
contains the parallel effect of the ara-C treatment of CHO cell
viability. The increasing concentrations of ara-C induce a pro
gressive increase in the specific activity of the template strands
from the DNA synthesized after the time of the ara-C pulse.
(The "Introduction"
contains the explanation of the rationale
for considering the relative specific activities of these template
strands as a measure of the relative extent of aberrant reinitia
tion of DNA replication induced by the ara-C pulse). The
relationship between the relative extent of aberrant reinitiation
and percentage of cell kill is illustrated in Chart 26, showing a
characteristic upwards tilting curve (similar in 3 different ex
periments). An asymptotic curve would be expected in this
graph since toxicity of ara-C is limited to those cells in S phase
at the time of drug treatment (2).
The following control experiment was performed in order to
determine whether this higher specific activity of template
strands from light-heavy DNA fraction could be trivially ex
plained by these strands having a higher specific activity the
lesser the proportion of DNA that was replicated in the pres
ence of BrdUrd. (The ara-C-treated cells under these condi
tions show a lower rate of DNA synthesis compared to the
control cells.) To examine the specific activity of the template
strands of the light-heavy DNA from cells in which lower pro
portions of DNA had been replicated, the same procedure as
with the control cells in Chart 1 was followed except that
portions of the control cell culture (no ara-C) were collected
after 1,2,3, and 4 hr in BrdUrd-containing medium (Chart 3).
Note that the shorter the BrdUrd-labeling period, the larger the
number of cells that were collected in order that approximately
equal amounts of DNA would be present in the light-heavy
fraction from each sample. The light-heavy DNA fractions were
again purified using 3 cycles of neutral CsCI gradients, and the
template and daughter strands were separated on alkaline
CsCI-Cs2SO4 gradients (Chart 3). The specific activities of the
template strands from the light-heavy DNA fraction relative to
the percentage of DNA replicated is presented in Chart 4. The
proportions of DNA replicated at successive time intervals after
BrdUrd addition were determined from the absorbance profiles
of the first cycle of neutral CsCI gradients (Chart 3, Row 7)
using the relationship:
% of DNA replicated = (LH/2) + (LH/2 + LL) x 100%
where LH was the amount of light-heavy DNA and LL was the
amount of light-light DNA. As can be seen in Chart 4, the
specific activities of template strands from the light-heavy DNA
fractions were unrelated to the proportion of DNA replicated.
(The specific activity of the first time fraction was 70.6 cpm/
/ig.) The percentage of DNA replicated in the ara-C experiment
illustrated in Charts 1 and 2 ranged from 19.6% for the control
to 3.8% for the cells treated with the highest concentration of
ara-C. In this latter experiment with cells without drug treatment
(Charts 3 and 4), the percentage of DNA replicated ranged
from 5.7% at 1 hr to 20.3% at 4 hr, a comparable range to that
observed in the ara-C experiment. Therefore, the higher spe
cific activities of this DNA fraction from cells treated with
progressively higher concentrations of ara-C (Charts 1 and 2)
cannot be due to the lower proportion of DNA replicated
following the ara-C pulse. The control experiment illustrated in
Charts 3 and 4 is one of 3 which gave comparable results. This
result also precludes the possibility that, under these experi
mental conditions, there are significant numbers of cells enter
ing a second S phase and replicating BrdUrd-containing
strands off 3H-labeled strands synthesized in the previous cell
cycle since, if this were the case, there would be a marked rise
with progressively longer times in the presence of BrdUrd of
the specific activity of the template strands in this light-heavy
DNA fraction. Whatever it is that results in some 3H label in the
[ara-C]
Chart 2. Relationship between aberrant DNA replication and loss of cell
viability in CHO cells following 2-hr pulses of a series of concentrations of ara-C.
In A, the specific activities (3H cpm//ig DNA) of the template strands from the
light-heavy DNA fractions of control and ara-C-pulsed cells from Chart 1 are
plotted against the concentration of ara-C used. The effects of these ara-C
concentrations on CHO cell viability assayed at the same time using plating
efficiency are also illustrated in A. The specific activities of the template strands
are expressed relative to the specific activity of those from the control cells. The
plating efficiencies are normalized to that of the control cells; bars, S.E. In B, the
relative specific activities of template strands from the light-heavy DNA fractions
are plotted directly against percentage of cell kill.
NOVEMBER 1982
template DNA strands under these experimental conditions, it
cannot be normal semiconservative DNA synthesis in a single
(or even a second) S phase. Instead, it must be some process
which is occurring in all of the cellular DNA at a constant rate
throughout the S phase.
In experiments to test whether ara-C and other DNA synthe
sis inhibitors induced DNA repair-type synthesis in CHO cells
[methodologically identical experiments to those presented in
Charts 2 and 4 of Woodcock and Cooper (34)], it was found
4747
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D. M. Woodcock et al.
B
D
Chart 3. A control experiment to determine
the effect the proportion of DMA replicated has
on the specific activity of the template strands
of the light-heavy DNA fraction. Asynchronously growing CHO cells were labeled with a
1-hr pulse of [3H]dThd. and the label was
chased for 1 hr in fresh medium, as in the
experiment illustrated in Chart 1. Cells were
then rinsed and incubated in medium contain
ing BrdUrd, FdUrd, and dCyd for 1 hr (Column
A). 2 hr (Column B), 3 hr (Column C), or 4 hr
(Column D). The light-heavy DNA fractions
were isolated from each sample using 3 cycles
of neutral CsCI gradients. Rows i to 3. suc
cessive steps in the isolation procedure. Bars,
fractions pooled at each step. Lett and right
arrows in each of the neutral CsCI gradients,
respectively, the CsCI densities of 1.75 and
1.70 g/ml. The strands of the purified lightheavy DNA fractions were separated and
banded in alkaline i:-.u c, . r.u. gradients
<
(flow 4). The bottoms of all gradients are to
the left. The fractions on the light density side
of those pooled from the light-heavy fraction
of the third cycle of neutral gradients (flow 3)
were filtered and counted. In all cases, any
remaining contamination of light-light DNA in
the light-heavy DNA fraction would have con
tributed an insignificant number of counts to
the alkaline density gradients.
10
20
1
10
20
1
10
20
1
10
20
fraction number
100
period of inhibition of DNA synthesis in CHO cells (with either
ara-C or methotrexate) caused a reduction in the amount of
this presumptive repair synthesis (not illustrated). This is con
sistent with the alternative explanation suggested previously
(34), namely, that this synthesis might be related to some form
of delayed ligation of DNA strands. This presumptive repair
synthesis is the most likely cause of 3H label in the template
50-
strands of the light-heavy DNA fraction in experiments such as
that illustrated in Charts 1 and 2. Since this apparent repair
synthesis is reduced following a period of DNA synthesis inhi
bition (34), it is most likely that any 3H label in the template
strands of this DNA fraction from ara-C-pulsed cells (as op
posed to 3H label present due to aberrant DNA synthesis)
1O
% DNA Replicated
20
Chart 4. Relationship between the proportion of DNA replicated and the
specific activities of the template strands from the light-heavy DNA fractions
prepared from cells incubated in BrdUrd-containing medium for 1, 2. 3, or 4 hr.
The specific activities (3H cpm//ig DNA) of the template strands in the alkaline
density gradients of Chart 3 are plotted against the proportion of DNA replicated
in BrdUrd-containing medium. The percentage of DNA replicated was calculated
from the absorbance profiles of the first cycle of neutral CsCI gradients in Chart
3 as described in the text.
that untreated CHO cells showed several times the level of
presumptive DNA repair synthesis that was apparent in the
DNA of other cell lines such as the Crow human cell line used
in these previous experiments (34) (not illustrated). (Presump
tive DNA repair synthesis is here defined as the incorporation
of nucleosides into regions of the chromosomal DNA not un
dergoing semiconservative replication at that time.) This incor
poration might be due to processes other than true repair
synthesis (34). As was observed with the Crow cell line (34), a
4748
would be less than that in the template strands of control cell
DNA. Consequently, the relative extent of aberrant DNA syn
thesis following ara-C treatment (Charts 1 and 2) is almost
certainly an underestimate of relative extent of this abnormal
replication. Hence, to summarize, this elevation of the specific
activities of this DNA fraction following ara-C treatment cannot
be explained by some cells entering a second S phase, by DNA
repair-type synthesis, or because of any inverse relationship
between the proportion of DNA replicated and the specific
activity of this DNA fraction.
Another explanation for presence of label in the template
strands from the light-heavy DNA fraction in these experiments
would be DNA recombinational and exchange events. For
instance, an elevated level of sister chromatid exchanges in
the untreated (control) CHO cells might possibly explain the
presence of 3H-labeled template strands. Using a different cell
line, we have previously presented direct experimental evi
dence against recombinational/exchange
events as an expla
nation for this aberrant form of DNA synthesis after ara-C
treatment (32). This experimental evidence was based on the
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VOL. 42
Aberrant DNA Replication and Loss of Cell Viability
requirement
that, for any DNA fragment
containing
3H and
BrdUrd in opposite strands to arise by DNA recombination or
exchange under these experimental conditions, these DNA
fragments must come from the actual site of exchange and that
that exchange must occur in a region in which, before the
exchange event occurred, there were adjacent 3H- and BrdUrdlabeled regions. The reasoning behind this requires extensive
discussion which is presented in full in Ref. 32. However, the
result of this is that under these experimental conditions, if the
DNA fragment size is doubled, this will approximately double
the specific activity of the template strands of the light-heavy
DNA fraction if the label in the template strands is present due
to DNA exchange events (32). The experimental procedure to
test whether recombinational or exchange events could explain
the 3H label in template strands of control and/or ara-C-treated
CHO cells was the same as in the experiment illustrated in
Chart 1 except that only control and 10~3 M ara-C (2 hr) treated
cultures were used. DNA was extracted and purified, one-half
of each sample was sheared with a 26-gauge needle which
gave DNA fragments of about 6,000 base pairs, and the other
one-half was sheared with a 20-gauge needle which gave
13,000 base-pair fragments (32). The light-heavy fraction was
purified from each sample of DNA and then banded in alkaline
CsCI-Cs2SO2 gradients as in Chart 1. The specific activities of
the template strands from these light-heavy DNA fractions are
presented in Table 2. The lack of effect of DNA fragment size
on the specific activities of this fraction from control and from
ara C-treated cells shows (a) that 3H label in template strands
of control cell light-heavy DNA is not due to recombinational
events and (o) that recombination cannot explain the elevated
specific activities of those template strands observed after araC treatment.
Another aspect of the results presented in Chart 1 that must
be noted is the dissimilarity of the profiles of 3H label in alkaline
density gradients of purified light-heavy DNA from untreated
(control) CHO cells and the profiles of this DNA fraction from
other cell lines used previously (Charts 1E and 3E of Ref. 34).
These latter charts show a peak of label in the daughter strands
at the bottom of the gradient with some label (but not enough
to form a distinct peak) at template strand density. By contrast,
the profile obtained with this DNA fraction from control CHO
cells (bottom left segment of Chart 1) shows peaks of compa
rable size at daughter strand and template strand positions.
The explanation for the appearance of sufficient counts at
template strand density to form a distinct peak in the latter
profile is most likely due to the several-times-higher
level of
presumptive repair synthesis in CHO cells relative to Crow cells
as discussed above. As to the label in daughter DNA strands,
Table 2
Effect of DNA fragment size on the specific activity of the template strands of
the light-heavy DNA fraction
The experimental protocol was the same as in the Chart 1 experiment except
that only control and 1CT3 M ara-C-treated cell cultures were prepared. One-half
of each DNA preparation was sheared to approximately 6,000 base pairs and
the other half to 13,000 base pairs before isolation of the light-heavy DNA
fraction from each and subsequent banding in alkaline CsCI-Cs SO, gradients.
Specific activity (3H cpm/jug DNA) of template
strands of the light-heavy DNA fraction
UNA iragment size (oase
pairs)6,000
13,000Control61.1
NOVEMBER 1982
M)268.3
(1 0'3
55.0ara-C
269.1
we have suggested previously that a major mechanism leading
to the appearance of 3H label at daughter strand density under
these experimental conditions was the interspersion of DMA
segments replicated at different times during the S phase
together with random shearing of DMA before density gradient
analysis resulting in 3H end labeling of BrdUrd-containing
daughter strands (32, 34, 35). Since CHO cells have replicons
which are on average more than twice the size of replicons in
the typical human cell (for review, see Ref. 5), it would be
expected that much less 3H label would be present due to this
mechanism in the BrdUrd-containing strands of the light-heavy
DNA fraction from CHO cells. These differences between CHO
cells and the human cell lines used previously can substantially
explain the difference in the appearances of the 3H label
profiles in the alkaline density gradients of the light-heavy DNA
fraction.
To test whether the relationship between cell killing and
aberrant DNA synthesis observed after an ara-C pulse was a
specific property of the ara-C molecule (or of inhibitors of DNA
polymerase in general) or whether it might be a general con
sequence of DNA replication forks being temporarily blocked,
DNA replication was inhibited indirectly with cycloheximide.
This compound is a highly specific inhibitor of mammalian
protein synthesis (20). Inhibition of protein synthesis has been
shown to cause a parallel and quantitively similar inhibition of
DNA synthesis which has been ascribed to the very small
¡ntracellular pools of DNA packaging proteins present in mam
malian cells (26). We have previously demonstrated that indi
rectly inhibiting DNA replication with cycloheximide also in
duces this aberrant form of DNA synthesis as is observed after
direct inhibition of DNA synthesis with ara-C or with ara-A (34).
Also, we demonstrated that a pulse of cycloheximide produces
S-phase-specific chromosome aberrations (31 ) as are induced
by a direct inhibitor of DNA synthesis like ara-C (17). These
observations are consistent with the reduction of clonogenicity
induced by a short pulse of this protein synthesis inhibitor
being due to the secondary block to DNA synthesis, in a
manner analogous to the effect produced by direct inhibition of
this process with a compound such as ara-C. An experiment
using CHO cells to examine simultaneously the relative extent
of cell killing and aberrant DNA replication after cycloheximide
treatment is illustrated in Charts 5 and 6. The experimental
protocol was the same as that used to examine the effect of
ara-C (Charts 1 and 2) except that the CHO cells were sub
jected to 2-hr pulses of cycloheximide at concentrations of 10,
100, or 1000 /xg/ml. The purification of the light-heavy DNA
fraction from control and cycloheximide-pulsed
cells and the
banding in alkaline CsCI-Cs2SO4 gradients of template and
daughter strands from this DNA fraction are illustrated in Chart
5. The relative specific activities of the template strands from
the light-heavy DNA fractions are illustrated in Chart 6A, to
gether with the results of the simultaneous assessment of the
effect of 2-hr pulses of the cycloheximide at these concentra
tions on the plating efficiencies of these cells. (In this experi
ment, the specific activity of the template strands from the lightheavy DNA fraction from control cells was 68.2 cpm/pg.) In
Chart 66, the relative extent of this aberrant DNA replication is
plotted directly against percentage of cell kill, producing a
similar type of relationship to that which was observed with
pulses of ara-C (Chart 2ß).This experiment illustrated in Charts
5 and 6 is one of the 4 experiments which produced compa4749
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D. M. Woodcock et al.
B
A
10
Chart 5. Estimating the relative extent of
aberrant DNA replication after 2-hr pulses of
cycloheximide. The experimental conditions
were the same as in Chart 1. Column A rep
resents the processing of DNA from control
CHO cells while Columns B, C, and D repre
sent processing of DNA from cells subjected
to 2-hr pulses of cycloheximide at 10. 100,
and 1000 fig/ml, respectively, flows 1 to 3,
successive steps in the 3 cycles of neutral
CsCI gradients used to purify the light-heavy
DNA fraction; Row 4, alkaline (_;•,<;!
C;; :;(.),
gradients of the purified light-heavy DNA frac
tions. The bottoms of all gradients are to the
left. Left and right arrows in each of the neutral
CsCI gradients, respectively, CsCI densities of
1.75 and 1.70 g/ml. Bars, pooled fractions.
The fractions on the light density side of those
pooled from the third cycle of neutral CsCI
gradients were filtered and counted. Any 'H
label contamination from light-light DNA pres
ent in the light-heavy DNA fractions would
have made only an insignificant contribution to
the counts in the alkaline gradients.
1005
E
WOO Q.
O
i
005-
05
JÃœL
„¿JWL.
wÃ-'W
O
400
200
.- •¿.. * /•
V
fraction
rabie results. The evidence discussed above, which strongly
suggests that the assessment of the relative extent of aberrant
DNA replication after ara-C treatment is only a minimal esti
mate, also applies equally well to the assessment of extent of
this process following a cycloheximide pulse. The asympto
matic nature of the relationship between cell kill and aberrant
DNA replication following a cycloheximide pulse would also be
expected since protein synthesis inhibitors have also been
shown to be S-phase specific in their cytotoxicity (2).
DISCUSSION
,.•-•.."...••
\
'
20
number
B
KX>-
5
io o
50
0-"-r
100
Direct inhibitors of DNA replication such as ara-C induce
chromosome aberrations in S-phase cells (17) and show Sphase-specific cytotoxicity (1, 2). Indirect inhibition of DNA
replication with protein synthesis inhibitors also induces Sphase-specific toxicity (2), and we have shown that, contrary
to some previous reports, the protein synthesis inhibitor cyclo
heximide also induces S-phase-specific chromosome damage
(31). The most likely hypothesis to explain the loss of cell
viability induced by a pulse of a drug such as ara-C is that it is
due to the resultant chromosome aberrations (1, 9). We have
also suggested that the S-phase-specific loss of clonogeneity
of mammalian cells induced by a pulse of a protein synthesis
inhibitor might similarly be due to the resultant S-phase-specific
chromosome damage (31, 34). Blocking DNA replication either
directly at the level of DNA polymerase with ara-C and 9-/6-Darabinofuranosyladenine
(6, 30) or indirectly by the lack of
proteins to package newly synthesized DNA (26) induces an
aberrant form of DNA replication (32,34,35). The most feasible
explanation for the nature of this aberrant replication is that it
represents aberrant reinitiation of DNA synthesis in DNA seg
ments which had already been replicated earlier in the same
cell cycle, resulting in some chromosomal DNA segments being
replicated more than once in a single cell cycle. We have
'*
f
20
20
20
4750
-500 «
[CYCLOHEXIMIDE]
WOO-
(/ug ml )
50
100
%CELL KILL
Chart 6. Relationship between aberrant DNA replication and loss of cell
viability in CHO cells following 2-hr pulses of a series of concentrations of
cycloheximide. In A, the specific activities (3H cpm/j<g DNA) of the template
strands from the light-heavy DNA fractions of control and cycloheximide-pulsed
cells from Chart 5 are plotted against the concentrations of cycloheximide used.
The effects of these concentrations of cycloheximide on CHO cell viability
assayed at the same time using plating efficiency are also illustrated in Chart 2A.
The specific activities of the template strands are expressed relative to the
specific activities of those from control cells. The plating efficiences are normal
ized to that of the control cells; bars, S.E. In Chart 68, the relative specific
activities of template strands from the light-heavy DNA fraction are plotted directly
against percentage of cell kill.
previously presented direct experimental evidence against sev
eral alternative explanations for this aberrant DNA synthesis
(32, 34, 35), and we have proposed mechanisms whereby
aberrant double replication of chromosomal DNA segments
would result in chromosome aberrations as well as defective
segregation of chromosomes at mitosis (33).
The method used previously to detect the occurrence of this
aberrant form of DNA replication has been modified to give an
estimate of the relative extent of this type of aberrant DNA
replication following increasingly severe treatments with both
a direct and an indirect inhibitor of DNA replication. (Note that
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RESEARCH
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VOL. 42
Aberrant DNA Replication
this methodology gives no absolute quantitative measure of the
extent of this abnormal replication.) Using CHO cells, the extent
of this aberrant DNA replication following 2-hr pulses of a
series of concentrations of ara-C and of cycloheximide were
determined together with simultaneous assessments of the
effects of these drug concentrations on cell viability. With both
ara-C and cycloheximide, it has been shown that a 2-hr pulse
of either drug induces a parallel increase in the extent of
aberrant DNA replication and loss of cell viability. This relation
ship between aberrant replication and cell killing gave similar
curves whether ara-C or cycloheximide was used to block DNA
replication (Charts 26 and 6B). While the data presented in this
paper do not constitute definitive proof of a causal relation
between this form of aberrant DNA replication and the induction
of chromosomal aberrations and cell death induced by these
agents, it is entirely consistent with this hypothesis.
Other explanations have been offered to explain cell killing
and chromosome damage induced by ara-C. Roberts et al. (22)
have used alkaline sucrose gradient analysis to demonstrate
that ara-C induces a decrease in the sedimentation rate of the
DNA of L1210 cells. The methodology used in this work re
sulted in highly anomalous patterns of sedimentation of the
cellular DNA (4, 22) which make the interpretation of the results
uncertain. Also, the conclusions of this paper are contrary to
findings previously published. In 2 studies, one using alkaline
sucrose gradient analysis (35) and one using alkaline elution
of DNA (24), ara-C treatment has been reported not to cause
a breakdown of the cellular DNA. If ara-C treatment results in
some DNA damage, then it is most likely that, after removal of
ara-C, some DNA repair synthesis would be apparent in the
cells. However, ara-C has been shown not to induce DNA
repair-type synthesis (34, 35), which also argues against araC inducing any direct damage to the cellular DNA.
It has also been suggested that the cytotoxicity of ara-C is
the direct consequence of the incorporation of ara-C into the
cellular DNA (13, 15) and that these incorporated ara-C moie
ties, in some unspecified way, lead to the subsequent death of
the cell. However, it has been shown that ara-G, the least
cytotoxic of the arabinose analogs of the DNA nucleoside
precursors, is also incorporated into DNA (18). In the case of
ara-G, incorporation into the cellular DNA appears to be a
relatively innocuous event in that the ara-G was shown to be
stably integrated into the DNA of L5178Y cells over 7.7 cell
doublings (18). While it is possible that there is some important
difference between the effect of ara-C and ara-G incorporation,
the very strong correlation between cytotoxicity and ara-C
incorporation into DNA might have another explanation other
than any directly toxic effect of the incorporated ara-C moieties.
ara-C has been shown to inhibit DNA replication by acting as
a leaky-chain terminator (30). Hence, the correlation between
the cytotoxicity and ara-C incorporation might arise through
the amount of this incorporation being a direct measure of the
relative time DNA replication forks remain stalled which, we
suggest, could be directly related to the probability of the
triggering of that process which results in the disruption of the
normal pattern of DNA replication and in aberrant double
replication of some chromosomal DNA segments.
Lymphocytes (as well as a number of cell lines of lymphoblastoid origin) show a phenomenon of rapid "interphase
death" following X-irradiation (7, 11, 14) or treatment with one
3 A. W. Harris and J. W. Lowenthal, personal communication.
NOVEMBER
1982
and Loss of Cell Viability
of a variety of cytotoxic drugs, including ara-C (23).3 Following
X-irradiation, the majority of cells from different tissues and the
majority of mammalian cell lines show a form of delayed
"mitotic death" (8, 11, 14). The CHO-K1 cell line used in the
experiments reported in this paper (and the human cell lines
such as G. K. and Crow cells used in previous publications)
exhibit a classic form of unbalance growth when incubated for
long periods (48 hr) with high concentrations (10~3 M) of araC, with cells greatly enlarging in volume but with there being
no appreciable loss of cells." Hence, the data presented in this
paper is relevant to the delayed form of cell death, not to rapid
"interphase death" which possibly represents a completely
different process. Thus, unless one is studying a cell type that
exhibits the phenomenon of rapid interphase death, the avail
able evidence is consistent with the hypothesis that the chro
mosome aberrations and cell killing induced by transiently
blocking the replication of the DNA of mammalian cells are due
to a resultant pathological derangement of the pattern of DNA
replication.
The aberrant form of DNA synthesis reported in this and in
previous publications (32-35) may well be important in explain
ing more than just the induction of chromosome damage and
cell killing by cytotoxic drugs which inhibit DNA synthesis.
Multiple rounds of DNA replication at one site in a chromosome
has been proposed by Botchan et al. (3) as a mechanism for
the production of free infectious copies of SV40 DNA in cells
which, before induction of viral DNA synthesis, contained single
stably integrated copies of the SV40 genome. In addition, the
small acentric chromosome fragments ("double minutes") as
sociated with methotrexate resistance in some mammalian cell
lines (10) may have also arisen through multiple rounds of
replication in random chromosome segments, this aberrant
replication having been induced by the methotrexate (another
inhibitor of DNA synthesis) used to select the resistant variants.
ACKNOWLEDGMENTS
We would like to thank Barbara Caldecoat for typing the manuscript and the
Medical Photography Department of the Cancer Institute for preparing the dia
grams.
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CANCER
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VOL. 42
Relationship between Aberrant DNA Replication and Loss of
Cell Viability in Chinese Hamster Ovary CHO-K1 Cells
David M. Woodcock, Jillian K. Adams and Ian A. Cooper
Cancer Res 1982;42:4744-4752.
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