[CANCER RESEARCH 41, 2483-2490, June 1981]
0008-5472/81 /0041-0000$02.00
Evidence for Double Replication of Chromosomal DMA Segments as a
General Consequence of DMA Replication Inhibition
David M. Woodcock1 and Ian A. Cooper
Haemato/ogy Research Unit, Cancer Institute, 481 Little Lonsdale Street, Melbourne,
ABSTRACT
We have previously presented evidence that a transient
inhibition of DNA synthesis by a pulse of 1-/J-D-arabinofuranosylcytosine (ara-C) results in a disruption of the pattern of
replication of the chromosomal DNA of cultured human cells,
resulting in some DNA segments being replicated more than
once in a single S phase. Further evidence is presented in this
paper that this effect is not a specific property of the ara-C
molecule in that a similar effect is produced in cells by a pulse
of 9-yS-D-arabinofuranosyladenine (ara-A) and also by a pulse
of cycloheximide. The activated form of ara-A and ara-C (the
triphosphates) both inhibit DNA synthesis at the level of the
polymerase. Double replication following an ara-A pulse dem
onstrates that double replication after an ara-C pulse is not
caused by some specific property of the ara-C molecule which
might be unrelated to any effect on DNA synthesis. However,
cycloheximide is an inhibitor of mammalian protein synthesis
and inhibits DNA synthesis only indirectly, probably through a
consequent deficiency of DNA-packaging proteins. Hence, the
occurrence of double replication of chromosomal DNA seg
ments following a pulse of cycloheximide is consistent with this
phenomenon being a general and nonspecific consequence of
the freezing of DNA replication forks.
INTRODUCTION
A transient inhibition of DNA synthesis in mammalian cells
results in chromosome aberrations (10, 12), cell death (4), and
even oncogenic transformation (1). A compound which is a
potent inhibitor of DNA synthesis but which has minimal effect
on RNA synthesis is ara-C2 (3, 4). ara-C exhibits S-phasespecific cytotoxicity of mammalian
icity is likely to be the consequence
induced by the ara-C treatment
induced chromosome aberrations
vious explanation since, following
cells (5), and this cytotoxof chromosome aberrations
(10, 11 ). However, ara-Cand cell death have no ob
a pulse of ara-C, cells re
cover their ability to synthesize new DNA chains and ligate
them to high molecular weight (10, 27), and also cells treated
with ara-C do not show degradation of their preformed DNA
(19, 28). Hence, more subtle events inside the cell must be
causing the chromosome damage and cell killing resulting from
a pulse of ara-C.
We have presented evidence that the mechanism controlling
the pattern of replication of the chromosomal DNA of cultured
'Recipient of a grant from the National Health and Medical Research Council
of Australia. To requests for reprints should be addressed.
2 The abbreviations used are: ara-C, 1-/î-D-arabinofuranosylcytosine; CHO,
Chinese hamster ovary; ara-A, 9-/3-p-arabinofuranosyladenine;
BrdUrd, 5-bromodeoxyuridine; FdUrd, 5-fluorodeoxyuridine;
dCyd, deoxycytidine; dGuo, deoxyguanosine; dThd, thymidine.
Received March 3, 1980; accepted February 13, 1981.
JUNE
1981
Victoria 3000. Australia
cells of human origin is disrupted following a pulse of ara-C,
resulting in some DNA segments being replicated more than
once in a single S phase (26, 28). These data indicated that,
following a pulse of ara-C, DNA strands were synthesized off
DNA template strands which had themselves been synthesized
only a few hr prior to the ara-C pulse. This can be visualized as
a partial endoreduplication of the chromosomal DNA occurring
within a single S phase. We have presented evidence against
a number of alternative explanations of this effect including
DNA repair synthesis, cells entering a second S phase, multiple
rounds of mitochondrial or Mycoplasma DNA synthesis, and
DNA recombinational and exchange events (26, 28). This effect
has been observed in 4 different mammalian cells lines. As well
as the 2 cell lines for which data were presented in previous
publications (26, 28), this effect has also been observed with
the K562 human leukemic cell line and with CHO cells.3
In this paper, we demonstrate that a similar effect on DNA
replication is also produced by a pulse of ara-A which, like araC, is a direct inhibitor of DNA polymerase (6) and also by a
pulse of cycloheximide, an inhibitor of protein synthesis (15,
17) which inhibits DNA synthesis by an indirect mechanism
probably related to its causing a deficiency in proteins neces
sary to package newly replicated DNA (20). Hence, we con
clude that disruption of the pattern of replication of the chro
mosomal DNA resulting in double replication of some chromo
somal DNA segments is a general and nonspecific conse
quence of a temporary interruption to DNA replication.
MATERIALS
AND METHODS
Cells and Culture Conditions. Experiments used Crow cells,
a human cell line derived from a retroorbital hemangioma in
the laboratory of Dr. T. R. Bradley (Cancer Institute, Melbourne,
Australia). Cells were grown in suspension culture in a-medium
supplemented with 10% fetal calf serum (Flow Laboratories,
Stanmore, New South Wales). Cells grew with a doubling time
of approximately 24 hr. Cell cultures were subdivided twice a
week.
Chemicals. Radiochemicals were purchased from the Radiochemical Centre, Amersham, United Kingdom. Nucleosides,
nucleoside analogs, cycloheximide, and Colcemid were pur
chased from the Sigma Chemical Co., St. Louis, Mo. All chem
icals were analytical reagent grade.
Isolation of Nuclei. Nuclei were isolated from whole cells by
the method of Lerner ef ai. (14).
Detection of DNA Repair Synthesis. The method used to
detect DNA repair synthesis was similar to that used previously
(28). BrdUrd (10 JIM), FdUrd (1 JIM), and dCyd (10 fiM) were
added to a culture of logarithmically growing Crow cells which
were allowed to continue DNA synthesis for 60 min to allow
3 D. M. Woodcock and I. A. Cooper, unpublished observations.
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D. M. Woodcock and I. A. Cooper
replicons synthesizing DNA at the beginning of the experiment
to have their replication completed in the presence of unlabeled
BrdUrd. At this point, inhibitor (either ara-A or cycloheximide)
was added to one-half of the culture (test cells), and 1 hr later,
these cells were resuspended in fresh medium containing 10
¡UM
BrdUrd, 1 ¡UM
FdUrd, and [3H]dGuo plus [3H]dCyd, both at
0.5 ¡uCi/ml.Control cells were transferred in this medium after
the 1 hr in the presence of unlabeled BrdUrd. Cells were
collected 4 hr after being placed in the radioactive medium. All
procedures were performed using minimal lighting, especially
of fluorescents. DNA extraction was performed as described
previously (26). The extracted DNA was dissolved in 0.15 M
NaCI:0.015 M trisodium citrate and further purified by treatment
with 50 /ig RNase-A per ml (Worthington Biochemical Corp.,
Freehold, N. J.) at 37°for 45 min, followed by 100 /ig pronase
per ml (Calbiochem, Carlingford, New South Wales; nucleasefree grade; self-digested) for a further 15 min. The aqueous
phase was then extracted with chloroform:octanol (24:1, v/v)
for 10 min at room temperature. After centrifugation, DNA was
precipitated from the resultant aqueous phase with cold
ethanol. CsCI isopyknic gradient fractionation of DNA was
performed as described previously (26). Before CsCI gradient
fractionation, DNA was sheared by 6 passages through a 26gauge needle which produced DNA fragments of about 6000
base pairs (26). The light-light DNA fraction from each DNA
sample was purified through 2 cycles of neutral CsCI gradients,
with 0.1 -ml aliquote being taken from each of the fractions from
the first round of neutral CsCI gradients (diluted previously with
0.5 ml of buffer for A26o readings) for the determination of 3H
content as described previously (28). The purified light-light
DNA was analyzed on alkaline CsCI gradients as described
previously (26, 28).
Detection of Double Replication. In this study, the method
used to detect aberrant double replication of DNA segments
was the second of the 2 methods described previously (26,
28). Each experiment used a logarithmically growing culture of
Crow cells which were labeled with [3H]dCyd at 0.5 ¡uCi/ml(22
odology to demonstrate double replication is used in the ex
periments reported in this paper. This method is based on the
basic nature of DNA semiconservative synthesis whereby, in a
given cell cycle, each of the strands of the parental DNA duplex
acts as a template for the synthesis of a daughter strand, with
the resultant replicated duplex containing one parental and one
daughter DNA strand. After a DNA segment is replicated in a
particular S phase, each resultant DNA duplex must contain
one parental and one newly synthesized strand. If a duplex is
present with a daughter strand opposite a daughter strand, it
could not be the product of normal semiconservative synthesis
in a single S phase. However, daughter-daughter
duplexes
could be formed by: (a) cells entering a second S phase; (to)
DNA repair synthesis; (c) DNA recombinational and exchange
events; (cOmultiple rounds of mitochondrial DNA synthesis; (e)
multiple rounds of Mycoplasma DNA synthesis; or ( f) abnormal
reinitiation of DNA synthesis in DNA segments already repli
cated earlier in that S phase, resulting in double replication of
those DNA segments. To demonstrate unequivocally that the
production of daughter-daughter duplexes was due to double
replication (Mechanism f), all other possible mechanisms must
be eliminated. To demonstrate the presence of daughterdaughter duplexes following a pulse of ara-C, cells were given
a pulse of [3H]dCyd followed by a 1.5-hr chase with a 2000fold excess of unlabeled dCyd. After the 1.5-hr chase period,
one-half of the cells was made 100 ¡UM
in ara-C and 1 hr later
transferred to fresh medium containing BrdUrd which resulted
in any DNA synthesized after the ara-C pulse being density
labeled. Control cells were placed directly into this medium
after the 1.5-hr chase. The light-heavy DNA fraction from
control and test cells was purified using 2 cycles of neutral
CsCI gradients. The purified light-heavy DNA which was the
DNA synthesized after the time of the ara-C pulse was disso
ciated, and the strands were analyzed on alkaline CsCI gra
dients. With the DNA from control cells, effectively all of the 3H
label was present at the density of the heavy strands, the
daughter strands which were synthesized after the time of the
addition of BrdUrd (26, 28). That any 3H label was present in
Ci/mmol) for 1 hr. The cells were transferred to fresh medium
containing 50 ¡UM
unlabeled dCyd for 1.5 hr to chase the [3H]- the heavy strand of this light-heavy DNA fraction of control
dCyd. After the 1.5-hr chase period, inhibitor was added to
cells might be due to (a) the chase procedure not being 100%
one-half of the culture (test cells). One hr later, test cells were
effective and (to) interspersion of DNA segments replicated at
transferred to fresh medium containing 10 /¿MBrdUrd, 1 JUM different times during S phase (9). Note that neither of these
FdUrd, and 10 ¿IM
dCyd. Control cells were placed directly into
mechanisms would result in nucleosides being incorporated
into a DNA template strand. However, when the light-heavy
the BrdUrd-containing medium after the 1.5-hr chase. If ColDNA from ara-C-pulsed cells was banded in alkaline CsCI
cemid was included in the protocol, it was added at 1 ¡ug/mlto
all media from the end of the 3H-labeling period onward. Cells
gradients, a high proportion (one-third to one-half) of the 3H
were collected from the BrdUrd-labeling medium at the times
label was present at the light-strand density, the density of the
template strand from which the BrdUrd-containing
daughter
indicated in the captions of the appropriate charts. DNA was
extracted as described above, and the light-heavy DNA frac
strand had just been synthesized (26, 28). In the case of araC, evidence has been presented that these 3H-labeled template
tions was purified through 2 cycles of neutral CsCI gradients.
The strands of the purified light-heavy DNA were dissociated
strands were not due to some cells entering a second S phase
since a similar distribution of 3H label was obtained whether or
and analyzed on alkaline CsCI gradients. Procedures were as
not Colcemid had been added to the medium to prevent mitosis
described previously (26).
Conceptual Basis of Method to Demonstrate Aberrant
(26). Nor was it due to mitochondrial DNA synthesis since
Double Replication. In previous publications (26, 28), we have similar results were obtained with DNA from whole cells or
presented evidence that, following a temporary inhibition of
isolated nuclei (26). Cells were routinely tested (21 ) and found
DNA synthesis by a pulse of ara-C, some DNA synthesis is to be free of Mycoplasma contamination. No evidence could
be found with the cell line used (GK cells) for DNA repair
reinitiated in DNA segments replicated earlier in that same S
synthesis following a pulse of ara-C under experimental con
phase, resulting in double replication of some segments of the
chromosomal DNA. Two types of experiments were presented
ditions where this aberrant form of DNA synthesis could be
in support of this hypothesis. The second more definitive meth
readily demonstrated (28). Also direct experimental evidence
2484
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VOL. 41
Aberrant DNA Replication after DNA Synthesis Inhibition
was presented against DMA recombinational and exchange
events being the explanation of these results (26). Hence, it
was concluded that the most feasible explanation for these
results was that, following the temporary interruption to DNA
synthesis due to the pulse of ara-C, the mechanism which
controls the sequence of replication of the chromosomal DNA
was disturbed, with some DNA synthesis being abnormally
reinitiated in DNA segments which had already been replicated
earlier in that S phase, resulting in aberrant double replication
of some DNA segments.
S
0.6
18
0.4
1.7
I
•-*
1.6
0.15
1.1
6.000
0.10
1.7
¡..000
0.05
1.6
2.000
0
0
1.9
IS
1.8
10
1.7
RESULTS
To test whether this effect was a peculiarity of the ara-C
molecule itself or was perhaps a more general phenomenon,
similar experiments were performed with ara-A. ara-A is a
purine nucleoside analog whereas ara-C is a pyrimidine analog.
The activated form of ara-A, like that of ara-C, inhibits DNA
replication directly at the level of the polymerase (6). Using the
experimental methodology as described above, logarithmically
growing Crow cells were labeled with a 1-hr pulse of [3H]dCyd,
and the label was chased for 1.5 hr with excess unlabeled
dCyd. After the chase period, ara-A at 0.5 mw was added to
one-half of the cells. One hr later, these cells were transferred
to fresh BrdUrd-containing medium. Crow cells after 1 hr in the
presence of 0.5 mM ara-A had [3H]dThd incorporation reduced
by more than 90% (not illustrated). The light-heavy DNA frac
tion from control and ara-A-pulsed cells were isolated and
purified through 2 cycles of neutral CsCI gradients, and the
strands of the purified light-heavy DNA fractions were sepa
rated and banded in alkaline CsCI gradients (Chart 1). As was
the case with ara-C (26, 28), a pulse of ara-A resulted in a high
proportion (approximately one-third) of the 3H label in the
isolated light-heavy DNA from the cells treated with inhibitor
banding as a peak at light-strand density (Chart 1F), the density
of the template strand from which the BrdUrd-containing strand
had been synthesized. This peak was absent in control
DNA (Chart 1E). In the experiment illustrated, Colcemid
jug/ml (to inhibit mitosis) had been included in all media
the end of the 3H-labeling period onward. Similar results
cell
at 1
from
have
also been obtained without the addition of Colcemid to the
experimental protocol (not illustrated).
Any 3H label in the daughter strands in the alkaline CsCI
gradients of Chart 1, £and F, could be present as noted above
because of the chase procedure not being totally effective as
well as through interspersion of DNA segments replicated at
different times during the S phase (9). The amount of this 3H
<o
N
-1
I
x
ca
TS
10
20
Fraction Number
Chart 1. Testing for aberrant reinitiation and double replication following a
pulse of ara-A. Logarithmically growing Crow cells were labeled with a 1-hr pulse
of [3H]dCyd, and the label was chased for 1.5 hr with a 2000-fold excess of
unlabeled dCyd. After the chase period. ara-A at 0.5 mw was added to one half
of the cells (test cells). One hr later, the test cells were transferred to fresh
medium containing 10 fiM BrdUrd. 1 fiM FdUrd. and 10 JIM dCyd. The other half
of the cells (control cells) was transferred directly to this medium after the 1.5-hr
chase. Control cells were incubated for 3 hr in this density-labeling medium and
test cells for 4 hr to allow a comparable proportion of DNA to be replicated in the
presence of BrdUrd in control and test cells. Colcemid (1 fig/ml) was present in
all media from the end of the 3H-labeling period onward. DNA was extracted and
purified as described in "Materials and Methods." The light-heavy DNA fraction
was purified through 2 cycles of neutral CsCI gradients, and the strands of the
purified light-heavy duplex were dissociated and separated in alkaline CsCI
gradients. A. and 6. initial neutral CsCI gradients; C and D. second cycle of
neutral CsCI gradients; E and F, alkaline CsCI gradients. A, C, and £.control cell
DNA; B, D. and F. test cell DNA. The fractions pooled from the neutral CsCI
gradients are indicated by the oars. The 10 fractions on the light-density side of
fractions pooled from the light-heavy peak from the second neutral CsCI gradients
were assayed for 3H content to check for contamination of the light-heavy fraction
with light-light DNA. Total cpm recovered from the alkaline CsCI gradients were
3896 in E and 2582 in F.
label is only a very small proportion of the total label incorpo
rated (see Chart 2 of Ref. 28). In order to characterize this 3H
longed low level of incorporation of [3H]nucleoside. To deter
mine the amount of unincorporated [3H]dThd remaining in the
label in the daughter strands and to see whether variations in
chase conditions and in the type of 3H-labeled nucleoside could
reduce the amount of 3H present in this fraction, the use of
[3H]dThd instead of [3H]dCyd was investigated. Cells were
labeled for 1 hr with 0.5 /iCi of [3H]dThd per ml and then
cells at increasing times after removal from the label, BrdUrd
(10 /UM)plus FdUrd (1 /IM) were added to portions of the culture
after 0.5, 1, and 1.5 hr in fresh medium. The cells were allowed
to synthesize DNA for 4 hr in the presence of BrdUrd before
being collected. As has been demonstrated to occur with other
mammalian cells (22), Crow cells show a preferential utilization
of dThd over BrdUrd since the light-heavy hybrid DNA pro
duced by these cells in the presence of FdUrd and BrdUrd plus
low levels of dThd was found to band in neutral CsCI gradients
at a lower density than would be expected from the molar ratio
of BrdUrd and dThd in the medium (not illustrated). Hence, 4
hr of DNA synthesis in the presence of BrdUrd and FdUrd
transferred to fresh medium without unlabeled nucleosides.
The use of excess unlabeled nucleosides as a chase procedure
was not used because of the inhibitory effect of excess dThd
on cell cycle progression (29) and because of the objection
that high levels of exogenous nucleosides might cause a large
expansion in the intracellular pool of dThd, diluting unincor
porated intracellular [3H]dThd and hence resulting in a pro-
JUNE 1981
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2485
D. M. Woodcock and I. A. Cooper
would have resulted in any remaining [3H]dThd being incorpo
method used, described in detail in "Materials
rated into the BrdUrd-containing
is similar to, but more sensitive than, the method used previ
ously to test for repair-type synthesis following a pulse of araC (28). The rationale for the method to detect DNA repair was
to test for the incorporation after treatment with a potentially
DNA-damaging agent of short patches of nucleosides into
regions of the DNA not involved in semiconservative replication
during the time of the experiments. These short patches of
nucleosides incorporated independently of replication were
taken as regions of presumptive DNA repair. Cells were incu
bated in medium containing unlabeled BrdUrd for 1 hr before
addition of 0.5 mw ara-A. After 1 hr in the presence of the ara-
daughter strands. DNA from
these cells was extracted and banded in alkaline CsCI gra
dients. The proportion of total [3H]dThd present in the heavy
strands progressively declined with time of chase, with 1.5 hr
of chase resulting in 0.58% of total cpm present in this region
of the gradient (Table 1). In attempts to reduce this apparent
residual incorporation of [3H]dThd, it was not possible to in
crease the rate of utilization of unincorporated intracellular
[3H]dThd by chasing in medium containing dialyzed fetal calf
serum since Crow
cell viability after
alyzed serum.
After [3H]dCyd
dCyd, the amount
cells showed growth retardation and loss of
even short periods in media containing di
labeling and chase with excess unlabeled
of [3H]dCyd present in heavy strands after
and Methods,"
A, cells were suspended in fresh medium containing BrdUrd
and [3H]dCyd plus [3H]dGuo. The control cells were transferred
chase procedure, this level was obtained after only 1 hr of
chase. Hence, we conclude that labeling with [3H]dCyd fol
directly into this medium following the 1 hr in the presence of
unlabeled BrdUrd. Cells were incubated initially in nonradioactive medium containing BrdUrd so that, after DNA extraction
and shearing of the DNA for banding in CsCI gradients, no DNA
fragments would be present which contained an interface be
tween a 3H-labeled BrdUrd-substituted region and an unlabeled
lowed by chase with excess unlabeled dCyd was superior to
that using [3H]dThd followed by a chase without unlabeled
nucleosides. The similar level of 3H label in daughter strands
unsubstituted region. The prior addition of unlabeled BrdUrd
ensured that, in effectively all fragments where there was an
interface between a 3H-labeled and unlabeled region in a DNA
after 1.5 hr of chase with both procedures would be consistent
with interspersion being the primary explanation of this appar
ent residual incorporation. However, about 0.5% of total label
incorporated may simply represent a practical lower limit of
efficiency of any chase procedure which will always be limited
by factors such as reincorporation of labeled nucleosides from
cells damaged during experimental manipulation. However,
neither interspersion or continued incorporation could intro
duce 3H label into template strands.
To ensure that the peak of 3H-labeled light strands from ara-
strand, both regions were BrdUrd substituted. [Note: DNA was
sheared to fragments of approximately 6000 base pairs (26).]
Hence, all [3H]nucleosides incorporated by normal semiconservative DNA replication would be present in the light-heavy
0.5, 1, or 1.5 hr of chase were similar to those obtained with
[3H]dThd (Table 1) with a level of 0.59% at 1.5 hr compared to
0.58% with [3H]dThd. However, with [3H]dCyd labeling and
A-pulsed cells was not present due to contamination of the
light-heavy DNA fraction with light-light DNA, the 10 fractions
on the light-density side of the fractions pooled from the lightheavy peak from the second cycle of neutral cell gradients
were assayed for 3H content (Chart 1C, control cell DNA; Chart
1D, ara-A-pulsed cell DNA). In both control and test cell
the light-heavy DNA was well separated from light-light
and the possible 3H label cross-contamination
between
DNA species would be too low to account for the amount
DNA,
DNA,
the 2
of 3H
label in template strands in the light-heavy DNA of ara-A-pulsed
cells.
Since DNA repair synthesis is the most feasible alternative
explanation of these results, these cells were tested for the
induction by a pulse of ara-A of DNA repair-type synthesis. The
Table 1
Comparison of 3H-labeling procedures
Cells were incubated with 0.5 nC\ of [3H]dThd or [3H]dCyd per ml for 1 hr.
Cells were then transferred to fresh medium. For the [3H]dCyd-labeled cells, this
medium contained 50 /IM unlabeled dCyd. At 0.5-hr intervals, BrdUrd (10 /IM)
and FdUrd (1 /IM) were added to portions of each culture. After 4 hr in the
presence of BrdUrd, cells were collected, and the DNA was extracted and
banded in alkaline CsCI gradients. In all gradients, total label recovered exceeded
10°cpm.
% of total 3H cpm in heavy strands
[3H)dCyd labeling with
Time of chase (hr)
[3H]dThd labeling
excess unlabeled dCyd
chase
0.5
1
1.5
1.13
0.72
0.58
1.02
0.57
0.59
2486
DNA fraction of the CsCI gradients, with none in
DNA which banded at the density of light-light
However, if DNA repair synthesis was occurring
DNA during the time the [3H]deoxynucleosides
these 3H-labeled nucleosides would be inserted
the bulk of the
duplex DNA.
in the cellular
were present,
into segments
of the chromosomal DNA not undergoing semiconservative
DNA replication at that time. Since the size of DNA repair
patches would be small compared to the sheared DNA fragment
size (8), the DNA duplexes containing regions of DNA repair
synthesis would still band at light-light duplex DNA density in
neutral CsCI gradients. The light-light duplex DNA from control
and ara-A-pulsed cells from an experiment as described above
was purified through 2 cycles of neutral CsCI gradients from
control and ara-A-pulsed cells, respectively (Chart 2). In the
initial neutral CsCI gradients (Chart 2, A and ß),the 3H cpm
were of 0.1 -ml samples taken from each fraction. (Each fraction
had been diluted previously with 0.5 ml of buffer for determi
nation of A26o's). Total cpm in each fraction would have been
approximately 7.5 times the values shown in Chart 2, A and B.
The purified light-light duplex DNA was dissociated, and the
separated strands were banded in alkaline CsCI gradients
(Chart 2, £and F; control and test, respectively) in order to
determine whether any 3H label had been incorporated into the
nonreplicating light-density strands due to short patches of
DNA repair synthesis. In the DNA of control cells (Chart 2£),
some presumptive DNA repair synthesis was present (3H label
at light-strand density). This presumptive DNA repair synthesis
was also present in DNA from ara-A-pulsed cells but to a lesser
extent (Chart 2F). The presence of repair-type synthesis in
control Crow cells is at variance with the lack of detectable
repair-type synthesis in untreated GK cells, another cell line of
human origin (28). It is also in disagreement with Gautschi ef
al. (8) who found no evidence for DNA repair synthesis in
untreated mammalian cells using a very similar methodology.
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VOL. 41
Aberrant DNA Replication after DNA Synthesis Inhibition
200.000-
0.0
1.0
0.4
tOOMO
17
0.2
0
sis. Hence, the result of experiments to demonstrate double
replication following a pulse of ara-A such as is presented in
O.I
«D
eg
parental DNA (7, 13). These patches of presumptive DNA
repair synthesis might thus be small regions of semiconservative replication involved in this delayed ligation. Moreover,
there is less of this presumptive repair synthesis in ara-Apulsed cells (Chart 2F) than control cells (Chart 2£).This would
be consistent with it being part of the process of semiconservative replication. However, whatever the true nature of this
apparent repair synthesis, that less is present after a pulse of
ara-A indicates that ara-A does not induce DNA repair synthe
0.4
1.7
0.2
•1.0
0
O.S
too
1.0
1.8
I). 4
50
0.3
1.7
0.2
0.1
10
20
20
Fraction Number
Chart 2. Testing for DNA repair synthesis after a pulse of ara-A. BrdUrd (10
/IM), FdUrd (1 /IM), and dCyd (10 JIM) were added to cultures of logarithmically
growing Crow cells. One hr later, ara-A at 0.5 rriM was added to one-half of the
cells (test cells). One hr later, these cells were transferred to fresh medium
containing BrdUrd, FdUrd, [3H]dCyd, and [3H]dGuo. The other half of the cells
(control cells) was placed directly into this medium after the 1 hr in the presence
of BrdUrd. Both control and test cultures were incubated for 4 hr in the Hlabeling medium before the cells were collected. DNA was extracted and purified
through 2 cycles of neutral CsCI gradients, and the strands of purified light-light
duplex DNA were dissociated and separated in alkaline CsCI gradients. A and B.
initial CsCI gradients; C and D, second cycle of CsCI gradients; £and F. alkaline
CsCI gradients. A, C, and E, control cell DNA; B, D, and F, test cell DNA.
Fractions pooled from the neutral CsCI gradients are indicated by the bars.
Aliquots of 0.1 ml from fractions of the initial neutral CsCI gradients were assayed
for 3H content (fractions diluted previously with 0.5 ml of buffer for A2K>deter
minations). The total cpm in each fraction of the initial neutral CsCI gradients
would have been approximately 7.5 times the cpm shown in A and B.
However, the methodology used in this study should be more
sensitive than that used in the 2 studies quoted above, since in
this case [3H]dCyd plus [3H]dGuo was used to label DNA repair
patches, neither of which nucleoside had to compete with
unlabeled BrdUrd for sites of incorporation as would be the
case with the [3H]BrdUrd or [3H]dThd used in the previous
studies. The peak of 3H label which is present in the light
strands of untreated Crow cells (Chart 2E) coincides with the
absorbance peak and shows no significant displacement to a
higher density. Since the DNA was sheared to approximately
6000 base pair fragments before any CsCI gradient fractionation, the patches of presumptive DNA repair synthesis could
have been at most a few hundred nucleotides long and prob
ably much shorter than that. An alternative explanation to its
being DNA repair synthesis is that it might be regions of delayed
semiconservative synthesis. There is evidence in eukaryotic
cells that there is a delay of some hours in the ligation of
semiconservatively replicated DNA to the molecular weight of
Chart 1 cannot be explained on the basis of it being an artifact
due to DNA repair.
Whether ara-C or ara-A is used in these experiments, DNA
replication is being inhibited directly at the level of DNA polymerase (6). To test whether aberrant reinitiation and double
replication is simply a general consequence of DNA replication
being temporarily halted, DNA replication was inhibited by an
indirect method.
Cycloheximide is a specific inhibitor of protein synthesis
acting at the level of the ribosome (17), inhibiting initiation and
translocation of the ribosomes along the mRNA (15). Addition
of the cycloheximide to mammalian cells results in a rapid
decrease in the rate of protein synthesis with a parallel and
quantitatively similar decrease in the rate of DNA synthesis
(20). Although the precise mechanism has not been definitely
demonstrated, it is likely that the parallel decrease in DNA
synthesis is due to lack of packaging proteins (both histones
and nonhistone chromosomal proteins) for the newly replicated
DNA (20). Hence, cycloheximide was considered to be a good
example of a compound producing an inhibition of DNA syn
thesis by an indirect mechanism.
In an experiment similar to that described above to demon
strate reinitiation and double replication after a pulse of ara-A,
Crow cells were labeled for 1 hr with [3H]dCyd, and the label
was chased for 1.5 hr with unlabeled dCyd. After the chase
period, cycloheximide at 100 /xg/ml was added to one-half of
the cells. One hr later, these cells were transferred to fresh
BrdUrd-containing medium. Control cells were transferred di
rectly to the BrdUrd-containing medium after the 1.5-hr chase.
A 1-hr treatment with cycloheximide at 100 jug/ml reduced the
incorporation by Crow cells of [3H]dThd into cold acid-insoluble
material by greater than 90% (not illustrated). The light-heavy
DNA fraction from control and cycloheximide-pulsed
cells was
purified through 2 cycles of neutral CsCI gradients, and the
strands of the isolated fractions of light-heavy duplex DNA
were dissociated and analyzed on alkaline CsCI gradients
(Chart 3). As was the case with ara-C and ara-A, a high
proportion (approximately one-half) of 3H label from the lightheavy DNA fraction banded as a peak at the density of the
template strands (Chart 3F), with this peak being absent from
the DNA of control cells (Chart 3£). Hence, this aberrant
reinitiation of DNA replication also occurs following inhibition
of synthesis by an indirect mechanism, namely, inhibition of
protein synthesis.
Again, to ensure that 3H label at template strand density was
not present due to contaminating light-light DNA in the lightheavy fraction, the 10 fractions from the second neutral CsCI
gradients (Chart 3, C and O) on the light-density side of the
samples pooled from the light-heavy peak were assayed for 3H
content. Again, the light-heavy fractions were well separated
JUNE 1981
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2487
D. M. Woodcock and I. A. Cooper
alkaline CsCI gradients (Chart 4£,light-light DNA from control
cells; Chart 4P, light-light DNA from cycloheximide-pulsed
cells). As was the case with the experiment illustrated in Chart
2, 3H label was present in the light-density strands from the
light-light DNA of control cells, indicating insertion of short
regions of 3H-labeled nucleotides into DNA segments not in
10.000
5.000
1
10
20
Fraction
1
10
Number
Chart 3. Testing for reinitiation and double replication following a pulse of
cycloheximide. Logarithmically growing Crow cells were labeled with a 1-hr pulse
of [3H]dCyd followed by a 1.5-hr chase with a 2000-fold excess of unlabeled
dCyd. After the chase period, cycloheximide (100 fig/ml) was added to one-half
of the cells (test cells). One hr later, the test cells were transferred to fresh
medium containing 10 JUMBrdUrd. 1 IÕMFdUrd, and 10 UM dCyd. The other half
of the cells (control cells) was transferred directly into this medium after the 1.5hr chase. Control cells were incubated for 3 hr in the density-labeling medium
and test cells for 4 hr to allow approximately equal proportions of DMA to be
replicated in the presence of BrdUrd in control and test cultures. Colcemid (1
/jg/ml) was present in all media from the time of the 3H-labeling period onward.
DNA was extracted and purified as described in "Materials and Methods." The
light-heavy DNA fraction was purified through 2 cycles of neutral CsCI gradients,
and the strands of the purified light-heavy duplex DNA were dissociated and
separated in alkaline CsCI gradient. A and B, initial neutral CsCI gradients; C and
D, second cycle of neutral CsCI gradients; E and F, alkaline CsCI gradients. A,
C, and £,control cell DNA; B, D. and F, test cell DNA. Fractions pooled from the
neutral CsCI gradients are indicated by the oars The 10 fractions on the lightdensity side of the fractions pooled from the light-heavy peak from the second
neutral CsCI gradients were assayed for 3H content to check for contamination
of the light-heavy fraction with light-light DNA. Total cpm recovered from the
alkaline CsCI gradients were 862 in £and 512 in F.
from the remaining light-light material (Chart 3, C and D).
As with ara-A, we tested whether a pulse of cycloheximide
induced DNA repair-type synthesis in Crow cells which might
have accounted for results such as those in Chart 3. Using the
same experimental method used with ara-A (Chart 2), Crow
cells were given a 1-hr pulse with 100 /¿gcycloheximide per
ml, and the light-light DNA fraction was purified through 2
cycles of neutral CsCI gradients. Aliquots of 0.1 ml were taken
from the first neutral CsCI gradients and assayed for 3H cpm.
The first neutral CsCI gradients are shown in Chart 4 (A, control
cell DNA; 8, cycloheximide-pulsed
cell DNA). The total 3H cpm
in each of the fractions would have been approximately 7.5
times the cpm shown on Chart 4, A and ß.The strands of the
purified light-light DNA fraction were separated and banded in
2488
volved in semiconservative synthesis during the time of the
experiment. As discussed above, this might be true DNA repair
synthesis, or it might be short regions of delayed ligation.
However, in cells given a pulse of cycloheximide, there is much
less of this presumptive DNA repair synthesis (Chart 4P) than
in the control cell DNA (Chart 4E). Again, the lower amount of
this presumptive repair synthesis following a period of reduced
DNA semiconservative synthesis would be consistent with its
being related in some way to semiconservative synthesis (i.e..
<o
M
0.50
0.25
10
20
10
20
Fraction Number
Chart 4. Testing for DNA repair synthesis after a pulse of cycloheximide.
BrdUrd (10 JIM), FdUrd (1 UM), and dCyd (10 ;uM) were added to cultures of
logarithmically growing Crow cells. One hr later, cycloheximide (100 fig/ml) was
added to one-half of the cells (test cells). One hr later, the test cells were
transferred to fresh medium containing BrdUrd, FdUrd. [3H]dCyd, and [3H]dGuo.
The other half of the cells (control cells) was placed directly into this medium
after the 1 hr in the presence of BrdUrd. Both control and test cultures were
incubated for 4 hr in the 3H-labeling medium before the cells were collected.
DNA was extracted as described in "Materials and Methods." The light-light
DNA fraction was purified through 2 cycles of neutral CsCI gradients, and the
strands of the purified light-light duplex DNA were dissociated and separated in
alkaline CsCI gradients. A and B, initial CsCI gradients; C and D, second cycle of
CsCI gradients; £and F, alkaline CsCI gradients. A, C, and £.control cell DNA;
B, D, and F, test cell DNA. Fractions pooled from the neutral CsCI gradients are
indicated by the bars. Aliquots of 0.1 ml from the fractions of the initial neutral
CsCI gradients fractions were assayed for 3H content (fractions diluted previously
with 0.5 ml of buffer for A26odeterminations). The total cpm in each fraction of
the initial neutral CsCI gradients would have been approximately 7.5 times the
cpm shown in A and B.
CANCER
RESEARCH
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VOL.
41
Aberrant DNA Replication after DNA Synthesis Inhibition
delayed ligation). In any case, the reduced extent of this
presumptive repair synthesis following a pulse of cycloheximide
argues against repair synthesis being the cause of the in
creased amount of 3H label present in template strands of DMA
duplexes replicated subsequent to a pulse of cycloheximide in
experiments such as those illustrated in Chart 3.
As was done previously with ara-C-pulsed cells (26), we
have determined the amount of double replication in DNA
extracted from whole cells and isolated nuclei following a pulse
of cycloheximide. There was quantitatively the same distribu
tion of 3H label between heavy and light strands of light-heavy
DNA extracted from whole cells or isolated nuclei from cycloheximide-pulsed cells (not illustrated). Hence, multiple rounds
of mitochondrial DNA synthesis cannot be a trivial explanation
of these results. Also cells were routinely monitored for Mycoplasma (21), obviating Mycoplasma
tion.
replication as an explana
DISCUSSION
We have previously presented data which indicates that,
following a pulse of ara-C, the mechanism controlling the
pattern of replication of the chromosomal DNA is disrupted,
resulting in some DNA segments being replicated more than
once in a single S phase (26, 28). This can be visualized as
some localized regions of the chromosomes undergoing endoreduplication in a single S phase. This paper presents evi
dence that the induction of double replication is not just a
peculiarity of the ara-C molecule itself but can also be induced
by transiently inhibiting DNA synthesis with ara-A and also with
cycloheximide. In the case of ara-C, we have specifically ob
tained evidence that this effect is not due to DNA recombinational and exchange events or to multiple rounds of DNA
replication in mitochondria or Mycoplasma (26, 28). In the
cases of ara-A and cycloheximide, we have tested whether this
double replication phenomenon might simply be an artifact due
to DNA repair synthesis. In both cases, we have found no
evidence for any stimulation of DNA repair-type synthesis fol
lowing a pulse of either inhibitor. The situation in relation to
repair synthesis is complicated by the increased sensitivity of
the method used in the present study to detect DNA repairtype synthesis. This has allowed the detection of what appears
to be DNA repair synthesis in previously unmanipulated control
cells (Charts 2 and 4). It is not unreasonable that there should
be a constant background of DNA repair in normal untreated
cells from spontaneous events at 37°.However, this presump
tive repair synthesis is reduced after DNA synthesis has been
transiently blocked by ara-A (Chart 2) or cycloheximide (Chart
4). This apparent repair synthesis is also reduced after a pulse
of ara-C. This effect has also been observed in 2 other cell
lines (K562 and CHO cells) as well as Crow cells.3 Since the
enzymes involved in DNA repair synthesis are in general less
sensitive to inhibitors than are the enzymes involved in semiconservative DNA replication (6), the observed reduction in the
amount of apparent repair synthesis after DNA replication
inhibition is more consistent with its being related to semiconservative replication than to repair. The explanation for this
apparent repair synthesis that we favor is that it is short regions
of normal semiconservative synthesis which is involved in de
layed ligation of replicated DNA to very high molecular weight
(7, 13). However, whatever the true explanation of this phe
nomenon, this apparent repair synthesis decreases after DNA
JUNE
synthesis inhibition. Hence, it cannot account for the increase
in 3H label from purified light-heavy DNA present at template
strand density in alkaline CsCI gradients in experiments testing
for double replication of chromosomal DNA segments (Charts
1 and 3).
As to other alternative explanations of the results shown in
Charts 1 and 3, cells were routinely screened for Mycoplasma
and were always found to be negative. As we have shown for
ara-C, the double replication after a pulse of cycloheximide
cannot be due to multiple rounds of mitochondrial DNA synthe
sis since similar results were obtained with DNA from whole
cells and from isolated nuclei. With the Crow cells used in these
experiments having a doubling time in logarithmic growth of
approximately 24 hr, the time of the experiment was too short
for cells in one S phase at the beginning of the experiment
(during the [3H]dCyd pulse) passing through G2, M, and Gt
during the time of the experiment. As an added precaution
against this possibility, Colcemid at 1 jug/ml was included in all
media from the time of the end of the [3H]dCyd pulse onwards.
It was not specifically tested experimentally as was done with
ara-C inhibition whether double replication after a pulse of araA or cycloheximide could have been due to DNA recombinational and exchange events. As has been fully argued previ
ously (26), the possibility that recombination or exchange could
explain results such as those in Charts 1 and 3 is extremely
remote. Hence, we conclude that the most feasible explanation
for the results presented in this paper is that double replication
of some chromosomal DNA segments is not just the result of
transiently inhibiting DNA synthesis by ara-C but also results
after similar levels of inhibition by ara-A and also by cyclohex
imide.
That this phenomenon occurs after a pulse of ara-A as well
as ara-C is not surprising since the activated forms of both
compounds (the triphosphates) act to inhibit DNA synthesis at
the level of the polymerase (6). However, what the result with
ara-A does show is that aberrant double replication after an
ara-C pulse is most likely mediated via the inhibition of DNA
synthesis and that the double replication is not the result of
some specific property of the ara-C molecule which might not
even have been related to any effect of 1-/S-D-arabinofuranosylcytosine triphosphate on DNA polymerase a (23). What is
more interesting is that double replication also occurs after
DNA synthesis is inhibited indirectly with cycloheximide due to
a consequent deficiency in the proteins necessary for pack
aging newly replicated DNA duplexes (20). We have suggested
that the double replication of some chromosomal DNA seg
ments following transient DNA synthesis inhibition by ara-C
may be the cause of the chromosome aberrations (28) and
hence of the cell death resulting from the pulse of ara-C (10,
11). Hence, an important test of this hypothesis is whether
other compounds which induce this double replication phenom
enon also induce chromosome aberrations and cell death, araA is a cytotoxic compound which has been shown to induce
chromosome aberrations in S-phase cells (16). However, araA is such a similar compound to ara-C as not to constitute a
particular stringent test of this hypothesis. However, the protein
synthesis inhibitor, cycloheximide, which affects DNA synthesis
only indirectly, constitutes a more appropriate test of this
model. If double replication following transient DNA synthesis
inhibition by ara-C is a major primary cause of chromosome
aberrations and subsequent S-phase-specific cytotoxicity, then
1981
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2489
D. M. Woodcock and I. A. Cooper
cycloheximide should also cause chromosome aberrations and
exhibit S-phase-specific cytotoxicity. It has already been shown
that protein synthesis inhibitors are cytotoxic to cells and that
this cytotoxicity is S-phase specific (5). Indeed, Bhuyan and
Frazer (3) have shown that streptovitacin A (acetoxycycloheximide) is not only S-phase specific but will protect cells in other
phases of the cell cycle from the toxicity of other S-phasespecific cytotoxics. S-phase cells were shown to be killed by
either the protein synthesis inhibitor or an S-phase-specific
agent such as ara-C while the protein synthesis inhibitor
stopped other cells from entering S-phase (3, 5). Hence, one
of our predictions of the consequences of double replication
following a cycloheximide pulse is already well documented.
However, for the second prediction, namely that this cycloheximide-induced double replication will cause chromosome
aberrations, we were unable to find any report of cycloheximide
inducing chromosome aberrations. On the contrary, there are
reports that cycloheximide is not clastogenic (2, 24). However,
in subsequent experiments with Crow cells, we have shown
that a 1-hr pulse of cycloheximide at 100 /ig/ml (the conditions
shown above to induce double replication) does in fact induce
chromosome damage (25). That the clastogenicity of cyclohex
imide was not detected previously was probably due to the
prolonged delay of cells with aberrations in reaching mitosis
(25). Hence, both of the predictions of our hypothesis as to the
consequences of double replication induced by a pulse of
cycloheximide have now been documented.
Therefore, from present evidence, we suggest that a tran
sient block to DNA replication, irrespective of the agent causing
this effect, disrupts the mechanism controlling the pattern of
replication of the chromosomal DNA, resulting in double repli
cation of some chromosomal DNA segments. Further, we sug
gest that this double replication induced in cells in S phase
results in chromosome aberrations and subsequent loss in cell
viability. We note that this hypothesis is entirely consistent with
the data of Ramseier ef a/. (18) who have shown a similar
decrease in cell viability of synchronized S-phase CHO cells
when DNA synthesis was inhibited to similar extents by hydroxyurea, FdUrd, excess dThd, or cycloheximide. In all cases,
almost total inhibition of DNA synthesis was necessary for large
reductions in cell viability with these authors suggesting that
the killing of S-phase cells was related to complete blocking of
DNA replication forks.
ACKNOWLEDGMENTS
We would like to thank Jill Adams for able technical assistance and Barbara
Caldecoat for typing the manuscript.
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RESEARCH
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VOL.
41
Evidence for Double Replication of Chromosomal DNA
Segments as a General Consequence of DNA Replication
Inhibition
David M. Woodcock and Ian A. Cooper
Cancer Res 1981;41:2483-2490.
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