evidence for double replication of chromosomal dma ... · dna repair synthesis, cells entering a...

[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, Victoria 3000. Australia

ABSTRACT

We have previously presented evidence that a transientinhibition of DNA synthesis by a pulse of 1-/J-D-arabinofurano-sylcytosine (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 thanonce in a single S phase. Further evidence is presented in thispaper that this effect is not a specific property of the ara-C

molecule in that a similar effect is produced in cells by a pulseof 9-yS-D-arabinofuranosyladenine (ara-A) and also by a pulseof cycloheximide. The activated form of ara-A and ara-C (the

triphosphates) both inhibit DNA synthesis at the level of thepolymerase. Double replication following an ara-A pulse demonstrates that double replication after an ara-C pulse is notcaused 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 synthesisand inhibits DNA synthesis only indirectly, probably through aconsequent deficiency of DNA-packaging proteins. Hence, the

occurrence of double replication of chromosomal DNA segments following a pulse of cycloheximide is consistent with thisphenomenon being a general and nonspecific consequence ofthe freezing of DNA replication forks.

INTRODUCTION

A transient inhibition of DNA synthesis in mammalian cellsresults in chromosome aberrations (10, 12), cell death (4), andeven oncogenic transformation (1). A compound which is apotent inhibitor of DNA synthesis but which has minimal effecton RNA synthesis is ara-C2 (3, 4). ara-C exhibits S-phase-

specific cytotoxicity of mammalian cells (5), and this cytotox-

icity is likely to be the consequence of chromosome aberrationsinduced by the ara-C treatment (10, 11 ). However, ara-C-

induced chromosome aberrations and cell death have no obvious explanation since, following a pulse of ara-C, cells re

cover their ability to synthesize new DNA chains and ligatethem to high molecular weight (10, 27), and also cells treatedwith ara-C do not show degradation of their preformed DNA

(19, 28). Hence, more subtle events inside the cell must becausing the chromosome damage and cell killing resulting froma pulse of ara-C.

We have presented evidence that the mechanism controllingthe 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-/Ã®-D-arabinofuranosylcytosine; CHO,

Chinese hamster ovary; ara-A, 9-/3-p-arabinofuranosyladenine; BrdUrd, 5-bro-modeoxyuridine; FdUrd, 5-fluorodeoxyuridine; dCyd, deoxycytidine; dGuo, de-oxyguanosine; dThd, thymidine.

Received March 3, 1980; accepted February 13, 1981.

cells of human origin is disrupted following a pulse of ara-C,

resulting in some DNA segments being replicated more thanonce 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 synthesizedonly a few hr prior to the ara-C pulse. This can be visualized as

a partial endoreduplication of the chromosomal DNA occurringwithin a single S phase. We have presented evidence againsta number of alternative explanations of this effect includingDNA repair synthesis, cells entering a second S phase, multiplerounds of mitochondrial or Mycoplasma DNA synthesis, andDNA recombinational and exchange events (26, 28). This effecthas been observed in 4 different mammalian cells lines. As wellas the 2 cell lines for which data were presented in previouspublications (26, 28), this effect has also been observed withthe K562 human leukemic cell line and with CHO cells.3

In this paper, we demonstrate that a similar effect on DNAreplication is also produced by a pulse of ara-A which, like ara-

C, is a direct inhibitor of DNA polymerase (6) and also by apulse of cycloheximide, an inhibitor of protein synthesis (15,17) which inhibits DNA synthesis by an indirect mechanismprobably related to its causing a deficiency in proteins necessary to package newly replicated DNA (20). Hence, we conclude that disruption of the pattern of replication of the chromosomal DNA resulting in double replication of some chromosomal DNA segments is a general and nonspecific consequence 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 inthe 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 timeof approximately 24 hr. Cell cultures were subdivided twice aweek.

Chemicals. Radiochemicals were purchased from the Radi-

ochemical Centre, Amersham, United Kingdom. Nucleosides,nucleoside analogs, cycloheximide, and Colcemid were purchased from the Sigma Chemical Co., St. Louis, Mo. All chemicals were analytical reagent grade.

Isolation of Nuclei. Nuclei were isolated from whole cells bythe method of Lerner ef ai. (14).

Detection of DNA Repair Synthesis. The method used todetect DNA repair synthesis was similar to that used previously(28). BrdUrd (10 JIM), FdUrd (1 JIM), and dCyd (10 fiM) wereadded to a culture of logarithmically growing Crow cells whichwere allowed to continue DNA synthesis for 60 min to allow

3 D. M. Woodcock and I. A. Cooper, unpublished observations.

JUNE 1981 2483

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D. M. Woodcock and I. A. Cooper

replicons synthesizing DNA at the beginning of the experimentto have their replication completed in the presence of unlabeledBrdUrd. 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 10Â¡UMBrdUrd, 1 Â¡UMFdUrd, and [3H]dGuo plus [3H]dCyd, both at

0.5 Â¡uCi/ml.Control cells were transferred in this medium afterthe 1 hr in the presence of unlabeled BrdUrd. Cells werecollected 4 hr after being placed in the radioactive medium. Allprocedures were performed using minimal lighting, especiallyof fluorescents. DNA extraction was performed as describedpreviously (26). The extracted DNA was dissolved in 0.15 MNaCI:0.015 M trisodium citrate and further purified by treatmentwith 50 /ig RNase-A per ml (Worthington Biochemical Corp.,Freehold, N. J.) at 37Â°for 45 min, followed by 100 /ig pronase

per ml (Calbiochem, Carlingford, New South Wales; nuclease-free 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 wasprecipitated from the resultant aqueous phase with coldethanol. CsCI isopyknic gradient fractionation of DNA wasperformed as described previously (26). Before CsCI gradientfractionation, DNA was sheared by 6 passages through a 26-

gauge needle which produced DNA fragments of about 6000base 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 with0.5 ml of buffer for A26oreadings) for the determination of 3H

content as described previously (28). The purified light-light

DNA was analyzed on alkaline CsCI gradients as describedpreviously (26, 28).

Detection of Double Replication. In this study, the methodused to detect aberrant double replication of DNA segmentswas the second of the 2 methods described previously (26,28). Each experiment used a logarithmically growing culture ofCrow cells which were labeled with [3H]dCyd at 0.5 Â¡uCi/ml(22

Ci/mmol) for 1 hr. The cells were transferred to fresh mediumcontaining 50 Â¡UMunlabeled dCyd for 1.5 hr to chase the [3H]-

dCyd. After the 1.5-hr chase period, inhibitor was added toone-half of the culture (test cells). One hr later, test cells were

transferred to fresh medium containing 10 /Â¿MBrdUrd, 1 JUMFdUrd, and 10 Â¿IMdCyd. Control cells were placed directly intothe BrdUrd-containing medium after the 1.5-hr chase. If Col-

cemid was included in the protocol, it was added at 1 Â¡ug/mltoall media from the end of the 3H-labeling period onward. Cells

were collected from the BrdUrd-labeling medium at the times

indicated in the captions of the appropriate charts. DNA wasextracted as described above, and the light-heavy DNA frac

tions was purified through 2 cycles of neutral CsCI gradients.The strands of the purified light-heavy DNA were dissociated

and analyzed on alkaline CsCI gradients. Procedures were asdescribed previously (26).

Conceptual Basis of Method to Demonstrate AberrantDouble Replication. In previous publications (26, 28), we havepresented evidence that, following a temporary inhibition ofDNA synthesis by a pulse of ara-C, some DNA synthesis is

reinitiated in DNA segments replicated earlier in that same Sphase, resulting in double replication of some segments of thechromosomal DNA. Two types of experiments were presentedin support of this hypothesis. The second more definitive meth

odology to demonstrate double replication is used in the experiments reported in this paper. This method is based on thebasic nature of DNA semiconservative synthesis whereby, in agiven cell cycle, each of the strands of the parental DNA duplexacts as a template for the synthesis of a daughter strand, withthe resultant replicated duplex containing one parental and onedaughter DNA strand. After a DNA segment is replicated in aparticular S phase, each resultant DNA duplex must containone parental and one newly synthesized strand. If a duplex ispresent with a daughter strand opposite a daughter strand, itcould not be the product of normal semiconservative synthesisin 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 exchangeevents; (cOmultiple rounds of mitochondrial DNA synthesis; (e)multiple rounds of Mycoplasma DNA synthesis; or ( f) abnormalreinitiation of DNA synthesis in DNA segments already replicated earlier in that S phase, resulting in double replication ofthose DNA segments. To demonstrate unequivocally that theproduction of daughter-daughter duplexes was due to double

replication (Mechanism f), all other possible mechanisms mustbe eliminated. To demonstrate the presence of daughter-daughter duplexes following a pulse of ara-C, cells were givena pulse of [3H]dCyd followed by a 1.5-hr chase with a 2000-

fold excess of unlabeled dCyd. After the 1.5-hr chase period,one-half of the cells was made 100 Â¡UMin ara-C and 1 hr later

transferred to fresh medium containing BrdUrd which resultedin any DNA synthesized after the ara-C pulse being density

labeled. Control cells were placed directly into this mediumafter the 1.5-hr chase. The light-heavy DNA fraction from

control and test cells was purified using 2 cycles of neutralCsCI gradients. The purified light-heavy DNA which was theDNA synthesized after the time of the ara-C pulse was disso

ciated, and the strands were analyzed on alkaline CsCI gradients. With the DNA from control cells, effectively all of the 3H

label was present at the density of the heavy strands, thedaughter strands which were synthesized after the time of theaddition of BrdUrd (26, 28). That any 3H label was present in

the heavy strand of this light-heavy DNA fraction of control

cells might be due to (a) the chase procedure not being 100%effective and (to) interspersion of DNA segments replicated atdifferent times during S phase (9). Note that neither of thesemechanisms would result in nucleosides being incorporatedinto a DNA template strand. However, when the light-heavyDNA from ara-C-pulsed cells was banded in alkaline CsCIgradients, a high proportion (one-third to one-half) of the 3H

label was present at the light-strand density, the density of thetemplate strand from which the BrdUrd-containing daughterstrand had just been synthesized (26, 28). In the case of ara-C, evidence has been presented that these 3H-labeled template

strands were not due to some cells entering a second S phasesince a similar distribution of 3H label was obtained whether or

not Colcemid had been added to the medium to prevent mitosis(26). Nor was it due to mitochondrial DNA synthesis sincesimilar results were obtained with DNA from whole cells orisolated nuclei (26). Cells were routinely tested (21 ) and foundto be free of Mycoplasma contamination. No evidence couldbe found with the cell line used (GK cells) for DNA repairsynthesis following a pulse of ara-C under experimental con

ditions where this aberrant form of DNA synthesis could bereadily demonstrated (28). Also direct experimental evidence

2484 CANCER RESEARCH VOL. 41



Aberrant DNA Replication after DNA Synthesis Inhibition

was presented against DMA recombinational and exchangeevents being the explanation of these results (26). Hence, itwas concluded that the most feasible explanation for theseresults was that, following the temporary interruption to DNAsynthesis due to the pulse of ara-C, the mechanism whichcontrols the sequence of replication of the chromosomal DNAwas disturbed, with some DNA synthesis being abnormallyreinitiated in DNA segments which had already been replicatedearlier in that S phase, resulting in aberrant double replicationof some DNA segments.

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 apurine 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 theexperimental methodology as described above, logarithmicallygrowing Crow cells were labeled with a 1-hr pulse of [3H]dCyd,

and the label was chased for 1.5 hr with excess unlabeleddCyd. After the chase period, ara-A at 0.5 mw was added toone-half of the cells. One hr later, these cells were transferredto fresh BrdUrd-containing medium. Crow cells after 1 hr in thepresence of 0.5 mM ara-A had [3H]dThd incorporation reduced

by more than 90% (not illustrated). The light-heavy DNA fraction from control and ara-A-pulsed cells were isolated and

purified through 2 cycles of neutral CsCI gradients, and thestrands of the purified light-heavy DNA fractions were sepa

rated and banded in alkaline CsCI gradients (Chart 1). As wasthe case with ara-C (26, 28), a pulse of ara-A resulted in a highproportion (approximately one-third) of the 3H label in the

isolated light-heavy DNA from the cells treated with inhibitorbanding as a peak at light-strand density (Chart 1F), the densityof the template strand from which the BrdUrd-containing strand

had been synthesized. This peak was absent in control cellDNA (Chart 1E). In the experiment illustrated, Colcemid at 1jug/ml (to inhibit mitosis) had been included in all media fromthe end of the 3H-labeling period onward. Similar results have

also been obtained without the addition of Colcemid to theexperimental protocol (not illustrated).

Any 3H label in the daughter strands in the alkaline CsCI

gradients of Chart 1, Â£and F, could be present as noted abovebecause of the chase procedure not being totally effective aswell as through interspersion of DNA segments replicated atdifferent times during the S phase (9). The amount of this 3H

label is only a very small proportion of the total label incorporated (see Chart 2 of Ref. 28). In order to characterize this 3H

label in the daughter strands and to see whether variations inchase conditions and in the type of 3H-labeled nucleoside couldreduce the amount of 3H present in this fraction, the use of[3H]dThd instead of [3H]dCyd was investigated. Cells werelabeled for 1 hr with 0.5 /iCi of [3H]dThd per ml and then

transferred to fresh medium without unlabeled nucleosides.The use of excess unlabeled nucleosides as a chase procedurewas not used because of the inhibitory effect of excess dThdon cell cycle progression (29) and because of the objectionthat high levels of exogenous nucleosides might cause a largeexpansion in the intracellular pool of dThd, diluting unincorporated intracellular [3H]dThd and hence resulting in a pro-

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Fraction NumberChart 1. Testing for aberrant reinitiation and double replication following a

pulse of ara-A. Logarithmically growing Crow cells were labeled with a 1-hr pulseof [3H]dCyd, and the label was chased for 1.5 hr with a 2000-fold excess ofunlabeled dCyd. After the chase period. ara-A at 0.5 mw was added to one halfof the cells (test cells). One hr later, the test cells were transferred to freshmedium containing 10 fiM BrdUrd. 1 fiM FdUrd. and 10 JIM dCyd. The other halfof the cells (control cells) was transferred directly to this medium after the 1.5-hrchase. Control cells were incubated for 3 hr in this density-labeling medium andtest cells for 4 hr to allow a comparable proportion of DNA to be replicated in thepresence of BrdUrd in control and test cells. Colcemid (1 fig/ml) was present inall media from the end of the 3H-labeling period onward. DNA was extracted andpurified as described in "Materials and Methods." The light-heavy DNA fraction

was purified through 2 cycles of neutral CsCI gradients, and the strands of thepurified light-heavy duplex were dissociated and separated in alkaline CsCIgradients. A. and 6. initial neutral CsCI gradients; C and D. second cycle ofneutral CsCI gradients; E and F, alkaline CsCI gradients. A, C, and Â£.control cellDNA; B, D. and F. test cell DNA. The fractions pooled from the neutral CsCIgradients are indicated by the oars. The 10 fractions on the light-density side offractions pooled from the light-heavy peak from the second neutral CsCI gradientswere assayed for 3H content to check for contamination of the light-heavy fractionwith light-light DNA. Total cpm recovered from the alkaline CsCI gradients were3896 in E and 2582 in F.

longed low level of incorporation of [3H]nucleoside. To determine the amount of unincorporated [3H]dThd remaining in the

cells at increasing times after removal from the label, BrdUrd(10 /UM)plus FdUrd (1 /IM) were added to portions of the cultureafter 0.5, 1, and 1.5 hr in fresh medium. The cells were allowedto synthesize DNA for 4 hr in the presence of BrdUrd beforebeing collected. As has been demonstrated to occur with othermammalian cells (22), Crow cells show a preferential utilizationof dThd over BrdUrd since the light-heavy hybrid DNA produced by these cells in the presence of FdUrd and BrdUrd pluslow levels of dThd was found to band in neutral CsCI gradientsat a lower density than would be expected from the molar ratioof BrdUrd and dThd in the medium (not illustrated). Hence, 4hr of DNA synthesis in the presence of BrdUrd and FdUrd

JUNE 1981 2485




would have resulted in any remaining [3H]dThd being incorpo

rated into the BrdUrd-containing daughter strands. DNA from

these cells was extracted and banded in alkaline CsCI gradients. The proportion of total [3H]dThd present in the heavy

strands progressively declined with time of chase, with 1.5 hrof chase resulting in 0.58% of total cpm present in this regionof the gradient (Table 1). In attempts to reduce this apparentresidual 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 cells showed growth retardation and loss ofcell viability after even short periods in media containing dialyzed serum.

After [3H]dCyd labeling and chase with excess unlabeleddCyd, the amount of [3H]dCyd present in heavy strands after

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 to0.58% with [3H]dThd. However, with [3H]dCyd labeling and

chase procedure, this level was obtained after only 1 hr ofchase. Hence, we conclude that labeling with [3H]dCyd fol

lowed by chase with excess unlabeled dCyd was superior tothat using [3H]dThd followed by a chase without unlabelednucleosides. The similar level of 3H label in daughter strands

after 1.5 hr of chase with both procedures would be consistentwith interspersion being the primary explanation of this apparent residual incorporation. However, about 0.5% of total labelincorporated may simply represent a practical lower limit ofefficiency of any chase procedure which will always be limitedby factors such as reincorporation of labeled nucleosides fromcells damaged during experimental manipulation. However,neither interspersion or continued incorporation could introduce 3H label into template strands.

To ensure that the peak of 3H-labeled light strands from ara-

A-pulsed cells was not present due to contamination of thelight-heavy DNA fraction with light-light DNA, the 10 fractionson the light-density side of the fractions pooled from the light-

heavy peak from the second cycle of neutral cell gradientswere assayed for 3H content (Chart 1C, control cell DNA; Chart

1D, ara-A-pulsed cell DNA). In both control and test cell DNA,the light-heavy DNA was well separated from light-light DNA,and the possible 3H label cross-contamination between the 2DNA species would be too low to account for the amount 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 theinduction by a pulse of ara-A of DNA repair-type synthesis. The

Table 1Comparison 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, thismedium 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 thepresence of BrdUrd, cells were collected, and the DNA was extracted andbanded in alkaline CsCI gradients. In all gradients, total label recovered exceeded10Â°cpm.

% of total 3H cpm in heavy strands

Time of chase (hr) [3H]dThd labeling

[3H)dCyd labeling with

excess unlabeled dCydchase

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method used, described in detail in "Materials and Methods,"

is similar to, but more sensitive than, the method used previously to test for repair-type synthesis following a pulse of ara-

C (28). The rationale for the method to detect DNA repair wasto test for the incorporation after treatment with a potentiallyDNA-damaging agent of short patches of nucleosides into

regions of the DNA not involved in semiconservative replicationduring the time of the experiments. These short patches ofnucleosides incorporated independently of replication weretaken as regions of presumptive DNA repair. Cells were incubated in medium containing unlabeled BrdUrd for 1 hr beforeaddition of 0.5 mw ara-A. After 1 hr in the presence of the ara-

A, cells were suspended in fresh medium containing BrdUrdand [3H]dCyd plus [3H]dGuo. The control cells were transferred

directly into this medium following the 1 hr in the presence ofunlabeled BrdUrd. Cells were incubated initially in nonradio-

active medium containing BrdUrd so that, after DNA extractionand shearing of the DNA for banding in CsCI gradients, no DNAfragments would be present which contained an interface between a 3H-labeled BrdUrd-substituted region and an unlabeled

unsubstituted region. The prior addition of unlabeled BrdUrdensured that, in effectively all fragments where there was aninterface between a 3H-labeled and unlabeled region in a DNA

strand, both regions were BrdUrd substituted. [Note: DNA wassheared to fragments of approximately 6000 base pairs (26).]Hence, all [3H]nucleosides incorporated by normal semicon-

servative DNA replication would be present in the light-heavy

DNA fraction of the CsCI gradients, with none in the bulk of theDNA which banded at the density of light-light duplex DNA.

However, if DNA repair synthesis was occurring in the cellularDNA during the time the [3H]deoxynucleosides were present,these 3H-labeled nucleosides would be inserted into segments

of the chromosomal DNA not undergoing semiconservativeDNA replication at that time. Since the size of DNA repairpatches would be small compared to the sheared DNA fragmentsize (8), the DNA duplexes containing regions of DNA repairsynthesis would still band at light-light duplex DNA density inneutral CsCI gradients. The light-light duplex DNA from controland ara-A-pulsed cells from an experiment as described abovewas purified through 2 cycles of neutral CsCI gradients fromcontrol and ara-A-pulsed cells, respectively (Chart 2). In theinitial neutral CsCI gradients (Chart 2, A and ÃŸ),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 determination 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, Â£and F; control and test, respectively) in order todetermine 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 2Â£),some presumptive DNA repair synthesis was present (3H label

at light-strand density). This presumptive DNA repair synthesiswas also present in DNA from ara-A-pulsed cells but to a lesserextent (Chart 2F). The presence of repair-type synthesis incontrol Crow cells is at variance with the lack of detectablerepair-type synthesis in untreated GK cells, another cell line ofhuman origin (28). It is also in disagreement with Gautschi efal. (8) who found no evidence for DNA repair synthesis inuntreated mammalian cells using a very similar methodology.





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Fraction NumberChart 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 logarithmicallygrowing Crow cells. One hr later, ara-A at 0.5 rriM was added to one-half of thecells (test cells). One hr later, these cells were transferred to fresh mediumcontaining 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 presenceof BrdUrd. Both control and test cultures were incubated for 4 hr in the H-labeling medium before the cells were collected. DNA was extracted and purifiedthrough 2 cycles of neutral CsCI gradients, and the strands of purified light-lightduplex DNA were dissociated and separated in alkaline CsCI gradients. A and B.initial CsCI gradients; C and D, second cycle of CsCI gradients; Â£and F. alkalineCsCI 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 assayedfor 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 gradientswould have been approximately 7.5 times the cpm shown in A and B.

However, the methodology used in this study should be moresensitive than that used in the 2 studies quoted above, since inthis case [3H]dCyd plus [3H]dGuo was used to label DNA repair

patches, neither of which nucleoside had to compete withunlabeled BrdUrd for sites of incorporation as would be thecase with the [3H]BrdUrd or [3H]dThd used in the previousstudies. The peak of 3H label which is present in the light

strands of untreated Crow cells (Chart 2E) coincides with theabsorbance peak and shows no significant displacement to ahigher density. Since the DNA was sheared to approximately6000 base pair fragments before any CsCI gradient fractiona-

tion, the patches of presumptive DNA repair synthesis couldhave been at most a few hundred nucleotides long and probably much shorter than that. An alternative explanation to itsbeing DNA repair synthesis is that it might be regions of delayedsemiconservative synthesis. There is evidence in eukaryoticcells that there is a delay of some hours in the ligation ofsemiconservatively replicated DNA to the molecular weight of

parental DNA (7, 13). These patches of presumptive DNArepair synthesis might thus be small regions of semiconserva-

tive replication involved in this delayed ligation. Moreover,there is less of this presumptive repair synthesis in ara-A-

pulsed cells (Chart 2F) than control cells (Chart 2Â£).This wouldbe consistent with it being part of the process of semiconser-

vative replication. However, whatever the true nature of thisapparent repair synthesis, that less is present after a pulse ofara-A indicates that ara-A does not induce DNA repair synthe

sis. Hence, the result of experiments to demonstrate doublereplication following a pulse of ara-A such as is presented in

Chart 1 cannot be explained on the basis of it being an artifactdue to DNA repair.

Whether ara-C or ara-A is used in these experiments, DNAreplication is being inhibited directly at the level of DNA polym-

erase (6). To test whether aberrant reinitiation and doublereplication is simply a general consequence of DNA replicationbeing temporarily halted, DNA replication was inhibited by anindirect method.

Cycloheximide is a specific inhibitor of protein synthesisacting at the level of the ribosome (17), inhibiting initiation andtranslocation of the ribosomes along the mRNA (15). Additionof the cycloheximide to mammalian cells results in a rapiddecrease in the rate of protein synthesis with a parallel andquantitatively similar decrease in the rate of DNA synthesis(20). Although the precise mechanism has not been definitelydemonstrated, it is likely that the parallel decrease in DNAsynthesis is due to lack of packaging proteins (both histonesand nonhistone chromosomal proteins) for the newly replicatedDNA (20). Hence, cycloheximide was considered to be a goodexample of a compound producing an inhibition of DNA synthesis by an indirect mechanism.

In an experiment similar to that described above to demonstrate 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 chaseperiod, cycloheximide at 100 /xg/ml was added to one-half of

the cells. One hr later, these cells were transferred to freshBrdUrd-containing medium. Control cells were transferred directly to the BrdUrd-containing medium after the 1.5-hr chase.A 1-hr treatment with cycloheximide at 100 jug/ml reduced theincorporation by Crow cells of [3H]dThd into cold acid-insoluble

material by greater than 90% (not illustrated). The light-heavyDNA fraction from control and cycloheximide-pulsed cells was

purified through 2 cycles of neutral CsCI gradients, and thestrands 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 highproportion (approximately one-half) of 3H label from the light-

heavy DNA fraction banded as a peak at the density of thetemplate strands (Chart 3F), with this peak being absent fromthe DNA of control cells (Chart 3Â£). Hence, this aberrantreinitiation of DNA replication also occurs following inhibitionof synthesis by an indirect mechanism, namely, inhibition ofprotein synthesis.

Again, to ensure that 3H label at template strand density wasnot present due to contaminating light-light DNA in the light-

heavy fraction, the 10 fractions from the second neutral CsCIgradients (Chart 3, C and O) on the light-density side of thesamples pooled from the light-heavy peak were assayed for 3H

content. Again, the light-heavy fractions were well separated

JUNE 1981 2487




10.000

5.000

1 10 20 1 10

Fraction NumberChart 3. Testing for reinitiation and double replication following a pulse of

cycloheximide. Logarithmically growing Crow cells were labeled with a 1-hr pulseof [3H]dCyd followed by a 1.5-hr chase with a 2000-fold excess of unlabeleddCyd. After the chase period, cycloheximide (100 fig/ml) was added to one-halfof the cells (test cells). One hr later, the test cells were transferred to freshmedium containing 10 JUMBrdUrd. 1 IÃ•MFdUrd, and 10 UM dCyd. The other halfof the cells (control cells) was transferred directly into this medium after the 1.5-hr chase. Control cells were incubated for 3 hr in the density-labeling mediumand test cells for 4 hr to allow approximately equal proportions of DMA to bereplicated 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 andseparated in alkaline CsCI gradient. A and B, initial neutral CsCI gradients; C andD, second cycle of neutral CsCI gradients; E and F, alkaline CsCI gradients. A,C, and Â£,control cell DNA; B, D. and F, test cell DNA. Fractions pooled from theneutral CsCI gradients are indicated by the oars The 10 fractions on the light-density side of the fractions pooled from the light-heavy peak from the secondneutral CsCI gradients were assayed for 3H content to check for contaminationof the light-heavy fraction with light-light DNA. Total cpm recovered from the

alkaline CsCI gradients were 862 in Â£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 mighthave accounted for results such as those in Chart 3. Using thesame experimental method used with ara-A (Chart 2), Crowcells were given a 1-hr pulse with 100 /Â¿gcycloheximide perml, and the light-light DNA fraction was purified through 2cycles of neutral CsCI gradients. Aliquots of 0.1 ml were takenfrom the first neutral CsCI gradients and assayed for 3H cpm.

The first neutral CsCI gradients are shown in Chart 4 (A, controlcell DNA; 8, cycloheximide-pulsed cell DNA). The total 3H cpm

in each of the fractions would have been approximately 7.5times the cpm shown on Chart 4, A and ÃŸ.The strands of thepurified light-light DNA fraction were separated and banded in

alkaline CsCI gradients (Chart 4Â£,light-light DNA from controlcells; Chart 4P, light-light DNA from cycloheximide-pulsed

cells). As was the case with the experiment illustrated in Chart2, 3H label was present in the light-density strands from the

light-light DNA of control cells, indicating insertion of shortregions of 3H-labeled nucleotides into DNA segments not in

volved in semiconservative synthesis during the time of theexperiment. As discussed above, this might be true DNA repairsynthesis, or it might be short regions of delayed ligation.However, in cells given a pulse of cycloheximide, there is muchless of this presumptive DNA repair synthesis (Chart 4P) thanin the control cell DNA (Chart 4E). Again, the lower amount ofthis presumptive repair synthesis following a period of reducedDNA semiconservative synthesis would be consistent with itsbeing related in some way to semiconservative synthesis (i.e..

<oM

0.50

0.25

10 20 10 20

Fraction NumberChart 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 oflogarithmically growing Crow cells. One hr later, cycloheximide (100 fig/ml) wasadded to one-half of the cells (test cells). One hr later, the test cells weretransferred to fresh medium containing BrdUrd, FdUrd. [3H]dCyd, and [3H]dGuo.

The other half of the cells (control cells) was placed directly into this mediumafter the 1 hr in the presence of BrdUrd. Both control and test cultures wereincubated 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 thestrands of the purified light-light duplex DNA were dissociated and separated inalkaline CsCI gradients. A and B, initial CsCI gradients; C and D, second cycle ofCsCI gradients; Â£and F, alkaline CsCI gradients. A, C, and Â£.control cell DNA;B, D, and F, test cell DNA. Fractions pooled from the neutral CsCI gradients areindicated by the bars. Aliquots of 0.1 ml from the fractions of the initial neutralCsCI gradients fractions were assayed for 3H content (fractions diluted previously

with 0.5 ml of buffer for A26odeterminations). The total cpm in each fraction ofthe initial neutral CsCI gradients would have been approximately 7.5 times thecpm shown in A and B.





delayed ligation). In any case, the reduced extent of thispresumptive repair synthesis following a pulse of cycloheximideargues against repair synthesis being the cause of the increased amount of 3H label present in template strands of DMA

duplexes replicated subsequent to a pulse of cycloheximide inexperiments 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 DNAextracted from whole cells and isolated nuclei following a pulseof cycloheximide. There was quantitatively the same distribution of 3H label between heavy and light strands of light-heavy

DNA extracted from whole cells or isolated nuclei from cyclo-heximide-pulsed cells (not illustrated). Hence, multiple rounds

of mitochondrial DNA synthesis cannot be a trivial explanationof these results. Also cells were routinely monitored for Myco-

plasma (21), obviating Mycoplasma replication as an explanation.

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 thanonce in a single S phase (26, 28). This can be visualized assome localized regions of the chromosomes undergoing en-doreduplication in a single S phase. This paper presents evidence that the induction of double replication is not just apeculiarity of the ara-C molecule itself but can also be inducedby transiently inhibiting DNA synthesis with ara-A and also withcycloheximide. In the case of ara-C, we have specifically obtained evidence that this effect is not due to DNA recombina-

tional and exchange events or to multiple rounds of DNAreplication in mitochondria or Mycoplasma (26, 28). In thecases of ara-A and cycloheximide, we have tested whether this

double replication phenomenon might simply be an artifact dueto DNA repair synthesis. In both cases, we have found noevidence for any stimulation of DNA repair-type synthesis following a pulse of either inhibitor. The situation in relation torepair synthesis is complicated by the increased sensitivity ofthe method used in the present study to detect DNA repair-

type synthesis. This has allowed the detection of what appearsto be DNA repair synthesis in previously unmanipulated controlcells (Charts 2 and 4). It is not unreasonable that there shouldbe a constant background of DNA repair in normal untreatedcells from spontaneous events at 37Â°.However, this presump

tive repair synthesis is reduced after DNA synthesis has beentransiently blocked by ara-A (Chart 2) or cycloheximide (Chart

4). This apparent repair synthesis is also reduced after a pulseof ara-C. This effect has also been observed in 2 other celllines (K562 and CHO cells) as well as Crow cells.3 Since the

enzymes involved in DNA repair synthesis are in general lesssensitive to inhibitors than are the enzymes involved in semi-conservative DNA replication (6), the observed reduction in theamount of apparent repair synthesis after DNA replicationinhibition is more consistent with its being related to semicon-servative replication than to repair. The explanation for thisapparent repair synthesis that we favor is that it is short regionsof normal semiconservative synthesis which is involved in delayed ligation of replicated DNA to very high molecular weight(7, 13). However, whatever the true explanation of this phenomenon, this apparent repair synthesis decreases after DNA

synthesis inhibition. Hence, it cannot account for the increasein 3H label from purified light-heavy DNA present at template

strand density in alkaline CsCI gradients in experiments testingfor double replication of chromosomal DNA segments (Charts1 and 3).

As to other alternative explanations of the results shown inCharts 1 and 3, cells were routinely screened for Mycoplasmaand were always found to be negative. As we have shown forara-C, the double replication after a pulse of cycloheximide

cannot be due to multiple rounds of mitochondrial DNA synthesis since similar results were obtained with DNA from wholecells and from isolated nuclei. With the Crow cells used in theseexperiments having a doubling time in logarithmic growth ofapproximately 24 hr, the time of the experiment was too shortfor 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 precautionagainst this possibility, Colcemid at 1 jug/ml was included in allmedia from the time of the end of the [3H]dCyd pulse onwards.

It was not specifically tested experimentally as was done withara-C inhibition whether double replication after a pulse of ara-A or cycloheximide could have been due to DNA recombina-

tional and exchange events. As has been fully argued previously (26), the possibility that recombination or exchange couldexplain results such as those in Charts 1 and 3 is extremelyremote. Hence, we conclude that the most feasible explanationfor the results presented in this paper is that double replicationof some chromosomal DNA segments is not just the result oftransiently inhibiting DNA synthesis by ara-C but also resultsafter 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 atthe level of the polymerase (6). However, what the result withara-A does show is that aberrant double replication after anara-C pulse is most likely mediated via the inhibition of DNA

synthesis and that the double replication is not the result ofsome specific property of the ara-C molecule which might noteven have been related to any effect of 1-/S-D-arabinofurano-

sylcytosine triphosphate on DNA polymerase a (23). What ismore interesting is that double replication also occurs afterDNA synthesis is inhibited indirectly with cycloheximide due toa consequent deficiency in the proteins necessary for packaging newly replicated DNA duplexes (20). We have suggestedthat the double replication of some chromosomal DNA segments following transient DNA synthesis inhibition by ara-C

may be the cause of the chromosome aberrations (28) andhence of the cell death resulting from the pulse of ara-C (10,

11). Hence, an important test of this hypothesis is whetherother compounds which induce this double replication phenomenon also induce chromosome aberrations and cell death, ara-

A is a cytotoxic compound which has been shown to inducechromosome aberrations in S-phase cells (16). However, ara-A is such a similar compound to ara-C as not to constitute a

particular stringent test of this hypothesis. However, the proteinsynthesis inhibitor, cycloheximide, which affects DNA synthesisonly indirectly, constitutes a more appropriate test of thismodel. If double replication following transient DNA synthesisinhibition by ara-C is a major primary cause of chromosomeaberrations and subsequent S-phase-specific cytotoxicity, then

JUNE 1981 2489




cycloheximide should also cause chromosome aberrations andexhibit S-phase-specific cytotoxicity. It has already been shown

that protein synthesis inhibitors are cytotoxic to cells and thatthis cytotoxicity is S-phase specific (5). Indeed, Bhuyan andFrazer (3) have shown that streptovitacin A (acetoxycyclohex-imide) is not only S-phase specific but will protect cells in otherphases of the cell cycle from the toxicity of other S-phase-specific cytotoxics. S-phase cells were shown to be killed byeither the protein synthesis inhibitor or an S-phase-specificagent such as ara-C while the protein synthesis inhibitorstopped other cells from entering S-phase (3, 5). Hence, oneof our predictions of the consequences of double replicationfollowing a cycloheximide pulse is already well documented.

However, for the second prediction, namely that this cyclo-heximide-induced double replication will cause chromosome

aberrations, we were unable to find any report of cycloheximideinducing chromosome aberrations. On the contrary, there arereports that cycloheximide is not clastogenic (2, 24). However,in subsequent experiments with Crow cells, we have shownthat a 1-hr pulse of cycloheximide at 100 /ig/ml (the conditions

shown above to induce double replication) does in fact inducechromosome damage (25). That the clastogenicity of cycloheximide was not detected previously was probably due to theprolonged delay of cells with aberrations in reaching mitosis(25). Hence, both of the predictions of our hypothesis as to theconsequences of double replication induced by a pulse ofcycloheximide have now been documented.

Therefore, from present evidence, we suggest that a transient block to DNA replication, irrespective of the agent causingthis effect, disrupts the mechanism controlling the pattern ofreplication of the chromosomal DNA, resulting in double replication of some chromosomal DNA segments. Further, we suggest that this double replication induced in cells in S phaseresults in chromosome aberrations and subsequent loss in cellviability. We note that this hypothesis is entirely consistent withthe data of Ramseier ef a/. (18) who have shown a similardecrease in cell viability of synchronized S-phase CHO cellswhen DNA synthesis was inhibited to similar extents by hy-droxyurea, FdUrd, excess dThd, or cycloheximide. In all cases,almost total inhibition of DNA synthesis was necessary for largereductions in cell viability with these authors suggesting thatthe 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 BarbaraCaldecoat for typing the manuscript.

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1981;41:2483-2490. Cancer Res David M. Woodcock and Ian A. Cooper InhibitionSegments as a General Consequence of DNA Replication Evidence for Double Replication of Chromosomal DNA

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