[CANCER RESEARCH 30, 2477—2483, October 1970J

SUMMARY

1-j3-D-Arabinofuranosylcytosine, an effective inhibitor ofDNA synthesis, induces chromatid breakage during the G1,5, and G2 phases of the mitotic cycle but not during Mphase in Don-C hamster fibroblasts. Breakage was preventedby simultaneous addition of deoxycytidine and ara-C in G1,5, and G2 . The maximum sensitivity to ara-C is during thelatter half of S and the beginning half of G2 . Deoxycytidineadded 30 min after ara-C decreased the number of chromosome breaks during the S phase but not during G1 or G2.

These results imply that the inhibition of DNA synthesis isintimately related to the genesis of chromatid breakage.Repair of these lesions require processes which are peculiarto S phase.

INTRODUCTION

ara-C4 (4) inhibits DNA synthesis in viral (8), animal (15),and tissue culture cells (17, 22, 27). There are at least 3molecular mechanisms responsible for this inhibition. Thecompound prevents the reduction of CDP (9), interferes withDNA polymerase (16, 23), and produces fraudulent macromolecules by incorporation into DNA and RNA polynucleotides (10, 11, 27). The relative importance of thesemechanisms or combination of mechanisms at the cellularlevel in producing the overall biological effect of ara-C is notknown.

Recently, considerable attention has been focused ondetermining the activity of a variety of antitumor agents onthe cell cycle in an effort to develop new approaches for

1This investigation was supported by USPHS Grants CA-01649 andCA-11050 and 1-493 from the American Cancer Society and waspresented in part at the Meeting of the Western Society for PediatricResearch, January 30, 1970.

2Present address: Department of Pediatrics, Albany Medical Center,Albany,N. Y. 12208.

3Leukemia Society of America Scholar. To whom reprint requestsshould be sent at Children's Hospital of Los Angeles, 4650 SunsetBlvd., Los Angeles,Calif.

4The abbreviations used are: am-C, 1@D-arabinofuranosylcytosine;CdR, deoxycytidine.

Received March 9, 1970; accepted June 8, 1970.

improving the clinical use of such compounds (26). Becauseof the effect of ara-C on DNA synthesis, the drug would beexpected to be a cell cycle-specific agent (7). Evidence for Sphase specificity has been obtained in vivo (28) and in vitro(18). Studies with the hamster fibroblast line, Don-C, asystem in which there is no appreciable metabolism of ara-C,have indicated a period of sensitivity of approximatelyone-half the generation time of 13 hr (18). This duration issimilar to that of S phase. Since ara-C is known to producechromosomal aberrations (2, 5 , 21 , 24), the effect of thedrug on the induction of chromosome breakage during thecell cycle was studied in an effort to define its locus ofaction more clearly.

MATERIALS AND METhODS

Cell Cultures. Cultures of hamster fibroblasts, clone Don-C,were obtained from Dr. Elton Stubblefield, M. D. AndersonHospital and Tumor Institute, Houston, Texas. They weregrown in stationary culture, with McCoy's 5A media (reconstituted from powder, Hyland Laboratories, Los Angeles,Calif.), supplemented with 20% fetal calf serum, penicillin,and streptomycin. The cells were fed daily and subculturedevery2 days.

The duration of the phases of Don-C cell cycle has beenmeasured previously (hr): G1, 3.9; 5, 6.2; G2, 2.2; M, 0.7(18). TG@ the generation time, was checked by growth curveanalysis every 4 to 6 weeks and remained consistentlybetween 12.5 and 13.1 hr.

The Effect of ara-C on the Cell Cycle. The cells, grown in8-oz prescription bottles, were exposed to ara-C at 1, 10,and 100 izg/ml for 2 hr and then harvested in a manner thatwould ensure that the cells were either in G2 or S. This wasaccomplished by exposing the cells to drug at 2 and 8 hrprior to harvest (G2 and 5, respectively) and then makingchromosome preparations. G1 -treated cells were obtained byexposure of 1 @.tg/miof ara-C 10 hr prior to harvest and 5iig/ml of ara-C 11.5 hr before harvest. Cells exposed to ara-C8 and 10 hr prior to harvest were treated with drug for 2 hr,the media were removed, and the cells were washed 3 timeswith Hanks' solution. Fresh nondrug-containing media wereadded for 6 and 8 hr, respectively , and Colcemid (CibaPharmaceutical Products, Inc., Summit, N. J.), 0.06 @ig/ml,was added 2 hr before harvest. Cells given 5 pg/mI of ara-C

2477OCTOBER 1970

Kinetics of 1*‘D-Arabinofuranosylcytosine-induced Chromosome1

William F. Benedict,2 Natalie Harris, and Myron Karon3

Laboratory of Cellular Phannacology, Division ofHematology, Department ofPediatrics, Children's Hospital of Los Angeles, and University ofSouthern CaliforniaSchool ofMedicine, Los Angeles, California90054

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The effect ofara C on chromosomebreaksNo.

of metaphases withindicatedPhaseara-C'@

(@g/ml)CdR(pg/ml)breaks/cell0 1—45—910+S0

11100

020046b

@ 0

29 19 244 6 07 22 110

00

10G2100

011

101000

00

20005

18 1047b 2 1

24 26 046 4 0

9 26 92 19 2117

0006

8M100049109G11

550

010050

0 018 28 345 5 00

10

Time topreparation

(h@)aNo.

of metaphases withindicatedbreaks/cellNo

CdRCdRb-01—4

5—910+01—45—910+1

2.511.5975432.521.525

25323537151522313523

223 018 014 111 230 328 226 219 015 00

200025000ND―

434744443622213335ND

ND7 03 06 06 013 125 126 316 115 0ND

000002000

William F. Benedict, Natalie Harris, and Myron Karon

11.5 hr before harvesting were treated in a similar manner,but washing was done 1 hr after exposure. The cellsharvested after 2 hr were treated simultaneously with ara-Cand Colcemid. Cells exposed to ara-C during M phase wereobtained by simultaneous treatment of cells with ara-C, 100pg/mI, and Colcemid for 30 mm. CdR, 20 jzg/ml, was alsoadded simultaneously to cultures treated with 1 @zg/mlofara-C in S and G2 . CdR, 100 .ig/ml, was added simultaneously to a culture treated with 5 pg/mI of ara-C in G1.

Table 1

EARLY S

LATE S

a@j@exposures of ara-C were br 2 hr except for M (30 mm) and Gi(1hi).5 j.@g/ml.

blndivaluai value performed at the sametime; control rate 48 ±2 (±1S.D.) for a total of 10 separate determinations performed at differenttimes.

Kinetic Studies. Duplicate cultures were exposed to ara-C,10 pg/mi, for 30 mm, at selected intervals prior to harvest,according to the design ifiustrated in Chart 1. The actualintervals used are shown in Table 2. Following drug exposure, both bottles of cells were washed 3 times withHanks' solution and refed, one bottle receiving growthmedia, the other growth media plus CdR, 100 jig/mi.Chromosome preparations were made following a 45-mmexposure to Colcemid, 0.06 jig/mI. One bottle of cellstreated with ara-C at 12 .5 hr and 11.5 hr was exposed toCdR for 2 hr and 1.5 hr respectively . The CdR was thenremoved, the cells were washed, and new media were added(Chart1, Table3). In a similarmanner,cellswereexposedto CdR for 1.5 hr after ara-C treatment 5 hr beforeharvesting.

As an alternative approach to kinetic analysis, replicatecultures were treated for 1 hr with ara-C, 10 jsgJml; the cellswere washed; and CdR, 100 j.zg/ml, was added at theintervals illustrated in Chart 2 and Table 4. Exposure toColcemid was for 45 mm.

The effectiveness of the washing procedure was tested byevaluating breakage following instantaneous washing after

The relationship ofchromosome breakage to cell cycle exposure time

aara@, 10 @zg/ml,for 30 min at times shown before harvest.bCdR, 100 @zg/ml, immediately after ara-C pulse and continued until

harvest.CNOt done.

exposure to ara-C, 10 jig/mI, and comparing this with ano-treatment control, as well as with 30-mm ara-C exposure(Table 5). These cells were exposed to drug for 33 hr priorto harvest and were consequently in early G2 or S.

Chromosome Preparations. Chromosome preparations weremade by a standard technique (6). The cells were washed 3times with Hanks' solution and trypsinized. The trypsinized

2478 CANCER RESEARCH VOL. 30

@CdR@

@CdR@

S@ G2 M

I I I2 K:@ 8 6 4 2 0

TIME TO PREPARATION (HR)

Qiart 1. Experimental design: cycle sensitivity to ara-C-inducedchromosome breakage. Following a 30-mis exposure to ara-C ( @),duplicate bottles were washed, and the nondrug-containing mediaadded. One-half the bottles CdR ( U). A scale denoting the durationof the cell cycle phases is superimposed for orientation. Colcemid wasadded 45 mm prior to harvest. This schema is diagrammatic and doesnot display all intervals used in the actual experiment tabulated inTable 2.

Table 2

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Cdi@CdR@S

&G2

M.‘.r

Kinetics of ara-C-induced Chromosome Breaks

-Si'.4

I— I I I U

8 6 4 2 0

TIME TO PREPARATION (HR)

Qiart 2. Experimental design: cycle sensitivity to CdR. nra-C, 10pg/ml, was added for 1 hr to replicate culture bottles. The cells werewashed, and CdR was added at 0, 2, and 5 hr following the wash,respectively. Colcemid was added 45 mm prior to harvest (arrow).The duration of 5, G2, and M are shown on the time axis fororientation.

cells were diluted with 0.9% NaCl solution and centrifugedat 1000 rpm in a PR-S clinical centrifuge at 4°.The cellbutton was resuspended in 0.95% sodium citrate for 20 mmwith gentle resuspension every 5 mm. The cells were thencentrifuged at 1000 rpm and the supernatant was removed.Fixation was performed by addition of freshly preparedmethanol:glacial acetic acid, 2: 1, for at least 20 mm. Thecells were centrifuged and resuspended in a volume suitablefor slide preparation. Slides were prepared by adding 3 dropsof the fixed cell suspension to a slide which had beenpreviously wetted with distilled water, and tI'e cell suspension was spread by blowing and quick drying with a blast ofwarm air. The slides were stained with Giemsa.

Fifty metaphase cells were counted to estimate chromatidand chromosome-type breakage and gaps. A gap was considered to be a lesion at least as wide as the width of thechromatid. A break was similar to a gap but at a differentangle than the adjacent intact chromatid arm (Fig. 1). Theslides were scanned with the lox objective of the microscope to identify metaphase plates that were sufficientlyspread. Chromosome breakage could not be seen at thismagnification. Once a suitable metaphase figure was identifled, the preparation was examined under the oil immersionlens for breakage. Only lesions that completely dissected thechromatids were counted, and these have all been reportedas “breaks―for the sake of simplicity.

Cell Passage Time. The rate of cell passage for 8 hrfollowing exposure to ara-C was determined in 2 ways. Thecell transit rate from G1 to S and from S to G2 wasevaluated by radioautographic methods (29). Simultaneously,the effect of this treatment on the mitotic index wasdetermined and compared with a control.

Replicate cell cultures growing on microscope slides in largeLeighton tubes were exposed to 10 jig/mI of ara-C for 30

OCTOBER 1970 2479

C,

Fig. 1. Metaphase showing chromosomes with large chromatid andisochromatid gaps (arrows). Inserted chromosome (right upper corner)shows a complete break as well as a gap. For simplicity, all suchlesions have been reported as “breaks.―

mm. The cells were washed 3 times with Hanks' solution.Replicate nondrug-treated cultures were handled in a similarfashion. Tritiated thymidine, 1 jiCi/mI (specific activity, 6.7Ci/mmole), was added, and the cultures were subsequentlytreated in 2 different ways. Total volume was 5 ml.

The effect of ara-C on continuous labeling with tritiatedthymidine was determined by harvesting the cultures atvarious intervals following drug treatment as indicated inChart 4. The 2nd group of cultures were pulsed withtritiated thymidine for 1 hr, and the cultures were harvestedat the same intervals.

The harvest procedure consisted of removing the microscope slides from Leighton tubes and washing them 3 timesin Hanks' media containing 10 jig/mi of nonradioactivethymidine. The cells were then fixed by immersing the slidesin methanol:acetic acid, 2: 1. Following fixation, the cellswere treated with 1 N perchloric acid at 4°for 20 mm andthen washed well with water. The slides were air dried andprepared for radioautography with Kodak NTB2 nucleartrack emulsion. The slides were stained with Giemsa. Mitoticindices were determined from the same slides. One thousandcells were counted under the oil immersion lens. Anyrecognizable phase of mitosis was scored.

Labeled Metaphases from Early S. Cultures growing in 8-ozbottles were pulse labeled for 30 mm with tritiated thymidine, 1 jiCi/ml, 8.5 hr prior to harvest. After washing, thecells were treated with 10 jig/nil of ara-C for 30 mm andwashed again, and fresh media were added. Colcemid wasadded 45 mm before harvesting. A replicate culture was

+4

@@1I@

*4@

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No. of metaphaseswithTime

prior―toharvestDuration

of CdRbexposureindicated

breaks/cell01—4

5—910+12.502523

2012.5223261011.5025230211.51.52920

015037112051.5500

00

13 12 II 10 9 8 7 6 5 4 3 2 I 0

William F. Benedict, Natalie Harris, and Myron Karon

treated identically except that tritiated thymidine and ara-Cwere added simultaneously 8 .5 hr prior to harvest.

Reagents. ara-C (NSC 63878) was obtained from theCancer Chemotherapy National Service Center, Bethesda,Md. Colcemid was obtained from Ciba. CdR was a productof Sigma Chemical Co., St. Louis, Mo. Tritiated thymidine(6.7 Ci/mmole) was from New England Nuclear Corp.,Boston Mass.

RESULTS

The Relation of Chromosome Breakage to the Phases ofthe Cell Cycle. The results of 2-hr exposure to various dosesof ara-C during G1 , 5, G2 , and M are illustrated in Table 1.In the absence of ara-C, 46/50 and 47/50 metaphases had nochromatid breaks. At 1 jig/mi, the number of cells withoutbreaks decreased to 29/50 and 24/50 in S and G2 , respectively. At 10 pg/ml of ara-C, the corresponding ratios ofmetaphases without breaks were 7/50 and 9/50 and, with100 jig/mI, the ratios were 5/50 and 2/50. Not only didmore chromatid breakage occur at higher dosage but also thenumber of metaphases showing 5 or more breaks increasedmarkedly. Cells exposed to 1 pg/ml of ara-C during G1showed no chromatid breakage, but with exposure to 5pg/ml for 1 hr only 18/50 metaphases were without breaks.Chromosome-type aberrations (dicentrics) were also found inmetaphases exposed to drug during G1 . The addition of CdRin a ratio of 20: 1 simultaneously with the addition of ara-C,prevented chromosome breakage.

Kinetic Studies. The effect on chromatid breaks of a30-mm “pulse―treatment with ara-C, 10 pg/ml, at varioustimes prior to cell harvest is ifiustrated in Table 2. Theserelationships are more clearly depicted in Chart 3 wherepercentage of metaphases with various numbers of breaks areplotted as a function of the time before preparation. Thepercentage of metaphases with breaks reaches its maximumtoward the end of S and the beginning of G2 . This isparticularly impressive for metaphases with 5 or morebreaks. The slight increase in the percentage of chromatidbreaks in G1 over that of early S is probably not significantand reflects the fact that the@ experiments were performed at a different time. Indeed, the most striking featureof the kinetic data (Table 2) is the relative consistency inthe number of breaks until 4 hr before harvest.

The addition of CdR 30 mm after exposure to ara-Creduced the frequency of breakage during S but not duringG2 . The addition of CdR for 1.5 hr in@ did not reducethe frequency of chromatid breaks, although exposure toCdR for the same period was effective in enhancing repair inS (Table 3).

The results of an alternate approach to evaluating theeffect of CdR addition on chromosome breakage is illustrated in Table 4. The number of breaks observed after a1-hr exposure to ara-C early in S is maximally diminishedwhen CdR is added immediately after exposure. The numberof breaks increases as CdR addition is delayed.

That the wash procedure was effective in removing absorbed drug is illustrated in Table 5 . The immediate institu

GI S G2 M

oA NO CdR

.@ CdR

ALL

80

70

@ 60

@ 50

g3@ 40

Ui 30

TIME TO PREPARATION (HR)

Chart 3. Relationship of sensitivity of chromosome breakage to thephases of the cell cycle. Plot of the data tabulated in Table 2. Arepresentation of the duration of the phases of the cell cycle issuperimposed for orientation. o—o , •—., percentage of metsphases with any breaks; t@—@, £—A, percentage of metaphaseswith 5 or more breaks. •, £, effect of CdR immediately followingwash. Colcemid, 0.06 Mg/ed, was added 45 mm before harvest. Thisexperiment follows the design illustrated in Chart 1. ALL, percentageof cells with at least 1 break.

Table 3

Effect ofshort exposure to CdR in G1 and S on repair

aaraC, 10 @&g/ed,added for 30 mm at the time indicated prior toharvest: 12.Sand 11.Shr,G1;5hr,S.

b@R, 100 @zg/ml,added immediately after 30 mm pulse of ara-C.

tion of the wash procedure after drug addition completelyprevented chromosome breakage, giving the same results asthe no-treatment control. Without the wash procedure, ara-Cinduced breakage in over 70% of the cells.

Cell Labeling. The results of the cell labeling studies areshown in Chart 4. The percentage of labeled cells increasedlinearly for 5.5 hr to a plateau of 80% labeling. Undersimilar conditions, untreated cells labeled to the extent of95% (18). The percentage of cells labeling after 1-hr pulse

2480 CANCER RESEARCH VOL. 30

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No. of metaphaseswithDuration

of CdRexposurePhaseindicated

breaks/cell01

—4 5—910+7EarlyS3316

105LateS2325023S—G21327460525

10 10

9

8

i::7z

a:w0.5

x

z—3

Effectiveness ofara-Cremoval by washing insituNature

ofcontrolNo.

of metaphases indicated withbreaks/cell0

1—4 5—910+Noara-C45

4 10ara-C'@15

27 25ara@Candwashb

46 4 0 0

Kinetics of ara-C-induced Chromosome Breaks

00

80

60

Table 4

Effect ofCdR on ara-C-induced breaks―

—JwO(/)

w.J—

‘U

U)I-z

0

CONTINUOUS

aara..t,, 10 isg/ml, for 1 hr, 8 hr prior to harvest. CdR, 100 pg/ed, untilharvest for the durations shown.

S.—,.G2

Table 52 4 6 8 10

TIME IN HOURS

Chart 4. Cell transit time was estimated following exposure ofreplicate cultures to ara-C, 10 pg/mi, for 30 mm. Following appropriate washing procedures, the cells were treated with tritiatedthymidine, 1 @Ci/ml.The curve of continuous labeling represents therate of accumulation of labeled cells. The degree of “pulse―labelingwas determined following a 1 h@exposure to tritiated thymidine (.).0- - - 0 , •-- -•, cell transit time. The percentage of cells progressing

from G1 to S was estimated by taking the difference between thepercentage of labeled cells at the time of sampling and the percentageof labeled cells at the beginning of the continuous labeling experiment. The rate of passage from S to G2 was determined bysubtracting the percentage of cells labeled by pulse exposure fromthose labeled by continuous exposure.

I 2 3 4 5 6 7 8 9 10

TIME IN HOURS

Chart 5. The mitotic indices were determined on replicate culturesgrown on microscope slides in Leighton tubes. ara-C-treated cells wereexposed to 10 @ig/mlof drug for 30 mm. Slides were fixed andstained with Giemsa and mitotic figures per 1000 cells determined.

received simultaneous tritiated thymidine and ara-C 8.5 hrprior to harvest, however, had poorly labeled or nonlabeledchromosomes. This difference reflects the inhibition of DNAsynthesis by ara-C.

a10 @g/odfor 30 mm, 3.5 hr prior to harvest.bAdthtion of 10 ,sg/mlfollowed immediately by wash and addition of

fresh median 3.5 hr prior to harvest.

exposure to tritiated thymidine varied from 55 to 60%. Theconsistency of these values indicated that treatment withara-C at 10 jig/ml for 30 mm produces no profound effectson labeling patterns.

The rate of cell transit from G1 to S and from S to G2 canbe estimated from the continuous labeling and pulse labelingcurves (29). By subtracting the percentage of labeled cellspresent during the 1st hr of continuous labeling from eachsubsequent increment, the passage rate from G1 to S can bedetermined. The passage rate from S to G2 can be estimatedby subtracting the percentage of pulse-labeled cells from thecurve of continuous labeling. These estimations are shown inChart 4.

There is a delay in the rate of transit from S to G@ for 3to 4 hr after drug treatment, whereupon the rate becomessimilar to that of G1 to S. After 6 hr, both rates decrease.

Mitotic Indices. The changes in mitotic index followingtreatment with ara-C compared to those in replicate culturesnot treated with ara-C are shown in Chart 5 . The m.itoticindex in the drug-treated cells is approximately two-thirds ofthat of the control.

At 7 hr, there is a rebound for both the control anddrug-treated cells.

Labeled Metaphases. All cells in metaphase pulsed 8 .5 hrprior to harvest with tritiated thymidine (early 5), andsubsequently treated with 10 jig of ara-C, contained labeledchromosome s. Consequently, all metaphases showingchromatid breakage were labeled. Cells in metaphase that

CONTROL

ara - C

2481OCTOBER 1970

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William F. Benedict, Natalie Harris, and Myron Karon

DISCUSSION

The induction of chromatid breakage by ara-C in the G1,5, and G2 phases of the cell cycle at essentially the samefrequency was not predicted by assuming an exclusive Sphase activity for ara-C (18). These findings prompted thekinetic experiments which indicated that the most sensitiveperiod of the cell cycle was the latter part of S and thebeginning of G2 . Chromatid aberrations were not foundduring M, indicating that ara-C has no direct effect onchromosomes even at a very high dose of drug, 100 jig/mI.This finding is of particular significance since M is a phase ofthe cell cycle when macromolecular synthesis is minimal(25). No breakage was observed in cells exposed to 1 jig/miof ara-C during the@ phase. However, 5 jig/mI of ara.C inG1 induced considerable chromatid breakage , as well aschromosomal-type lesions which are characteristic of thoseproduced in@ . ara-C-induced chromosomal-type aberrationshave also been reported in cultures of human leukocytestreated with phytohemagglutinin and serum (4, 5). Sincechromatid breakage occurs in all phases of the cell cycleexcept M, the breakage cannot be fully explained on thebasis of inhibition of DNA synthesis alone. Ahnstrom andNatarajan (1) have suggested that breakage in G1 and G2 isdue to factors favoring DNA degradation by DNA polymerase such as a decrease in the triphosphate pool. Such adecrease would result from blockage of dCDP or dCTPproduction by ara-C.

An important aspect of ara-C-induced chromosome breakage is the effect of CdR. This compound is known to reversethe lethal effect of ara-C when administered simultaneouslywith or shortly after the nucleoside. Simultaneous administration of ara-C and deoxycytidine in a ratio of 1:20 prevented chromatid breakage in Don-C cells. The effect of delayed addition of CdR was dependent upon the phase of thecell cycle in which the nucleoside was added. If the addition ofCdR was delayed for as little as 30 mm in@ or G2 , noreversibility of chromosome breakage was observed. On theother hand, if CdR was added early in 5, considerablereversibility was observed. The ability of CdR to decreasechromatid breakage diminished as the cell approached G2.

The CdR results imply that ara-C may not only inducechromatid breaks by interference with DNA synthesis or thefavoring of DNA degradation (1) but may also interfere withDNA repair (12). This dual role would be consistent with therecent findings that DNA polymerase, which is inhibited byara-C (16, 23), is involved in both the synthesis and repair ofDNA (20).

An additional factor in the mechanism of ara-C-inducedchromatid breakage is discontinuity in the associated histone.Protein synthesis rather than DNA or RNA synthesis appearsto be necessary for rejoining chromosome breaks (30).Histone synthesis is coupled to that of DNA. It is inhibitedby ara-C and occurs during the S phase (3 , 13). This suggeststhat the inability of CdR to reduce chromatid breakage inG1 and G2 might be due to an absence of histone synthesisleading to failure to “repair―the break with protein.

ara-C is known to affect cell transit time primarily byslowing the rate of passage from S to G2 (19). This effect is

dose related. A significant slowing in transit time couldcomplicate the interpretation of the kinetic data. That thiswas not the case for a 30-mm exposure to 10 jig/mi isindicated by study of the mitotic indices (Chart 5), by theestimations of cell transit times from continuous and pulseexperiments (Chart 4), and by the fact that all metaphasesexhibiting chromatid breaks were labeled 8 .5 hr after a pulseexposure to tritiated thymidine followed by a 30-mm treatment with ara-C. If a significant delay in transit from S toG2 in the presence of a normal@ to S transit hadoccurred, the mitotic indices should have fallen progressivelyby 3 hr as compared to the control. Similarly, there wouldhave been no recovery in the rate of S to G2 transit seen inChart 4. In addition, chromatid breakage would have occurred in unlabeled metaphases.

The lethality of ara-C in terms of decreased plating efficiency can be correlated with the induction of 5 or morechromatid breaks per cell (M. Karon, W. F. Benedict, N. Harris,and P. Saarinew, in preparation). This means that the mostsensitive period for a given dose of ara-C is the latter half of Sand the first half of G2 , a period of approximately 5 hr. Theseresults are consistent with the evidence for enhanced sensitivityduring S phase obtained by plating efficiency (18). The lattermethod was not sensitive enough to demonstrate the effect ofara-C on early G2 and G1.

Since ara-C can induce chromatid breakage in G1 , S, andG2 and since these lesions can be prevented by simultaneousaddition of CdR, the interruption of DNA synthesis must beintimately related to chromatid breakage. The CdR reversalstudies indicate that once breakage has been induced, repaircan occur only during S phase. The relationship of thesephenomena to histone synthesis remains to be elucidated.

ACKNOWLEDGMENTS

We acknowledge the expert technical assistance of Mrs. Vivian Bettsand Mrs. Joan Scher.

REFERENCES

1. Ahnstrom, C., and Natarajan, A. T. Mechanism of ChromosomeBreakage—aNew Theory. Hereditas, 54: 379—388,1966.

2. Bell, W. R., Whang, J. J., Carbone, P. P., Brecher, G., and Block,J. B. Cytogenetic and MorphologicAbnormalities in Human BoneMarrow Cells during Cytosine Arabinoside Therapy. Blood, 27:771—781, 1966.

3. Borun, T. W., Scharff, M. D., and Robbins, E. Rapidly Labeled,Polyribosome-associated RNA Having the Properties of HistoneMessenger. Proc. Natl. Acad. Sd. U. S., 58: 1977—1983, 1967.

4. Brewen, J. G. The Induction of Chromatid Lesions by CytosineArabinoside in Post-DNA Synthetic Human Leukocytes. Cytogenetics, 4: 28—36,1965.

5. Brewen, J. G., and Christie, N. 1. Studies on the Induction ofChromosomal Aberrations in Human Leukocytes by CytosineArabinoside. Exptl. Cell Res., 46: 276—291, 1967.

6. Brown, C. D., and Porter, I. H. Methods in Cytogenetics. ZeissMetteilung.,48:330—375,1968.

7. Bruce, R. W., and Valeriote, F. A. Normal and Malignant StemCells and Chemotherapy. In: The Proliferation and Spread ofNeoplastic Cells, Univ. of Texas, M. D. Anderson Hospital andTumor Institute at Houston, pp. 409—422.Baltimore: TheWilliams & Wilkins Co., 1968.

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Kinetics of ara-C-induced Chromosome Breaks

8. Buthala, D. A. Cell Culture. Studies on Antiviral Agents: I.Action of Cytosine Arabinoside and Some Comparisons with5-Iodo-2.deoxyuridine. Proc. Soc. Exptl. Biol. Med., 115: 69—77,1964.

9. Chu, M. Y., and Fischer, G. A. A Proposed Mechanismof Actionof 1-j3-D-Arabinosylcytosineas an Inhibitor of the Growth ofLeukemic Cells. Biochem. Pharmacol., 11: 423—430, 1962.

10. Ow, M. Y., and Fischer, G. A. Comparative Studies of LeukemicCells Sensitive and Resistant to Cytosine Arabinoside. Biochem.Pharmacol.,14:333—341,1965.

11. Chu, M. Y., and Fischer, G. A. The Incorporation of 3H-CytosineArabinoside and Its Effects on Murine Leukemic Cells(L-5178Y). Biochem. Pharmacol., 17: 752—767,1968.

12. Qeaver, J. E. Repair Replication and Degradation of Bromouracil-substituted DNA in Mammalian Cells after Irradiation withUltra-violet Light. Biophys. J., 8: 775—791, 1968.

13. Das, N. K., and Alfert, M. Cytochemical Studies on the Concurrent Synthesis of DNA and Histone in Primary Spermatocytes ofUrechis caupo. Exptl. Cell Res., 49: 51—58,1968.

14. Evans, J. S., and Mengel, G. D. The Reversal of CytosineArabinoside Activity in Vivo by Deoxycytidine. Biochem.Pharmacol., 13: 989—994, 1964.

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2483OCTOBER 1970

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1970;30:2477-2483. Cancer Res   William F. Benedict, Natalie Harris and Myron Karon  Chromosome Breaks

-d-Arabinofuranosylcytosine-inducedβKinetics of 1-

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